r~ INSTITUT PASTEUR/ELSEVIER Paris 1990

Res. Virol.

,,,,90, 141,

LJT--LUJ

REGULATION OF HIV1 REPLICATION IN P R O M O N O C Y T I C U937 C E L L S

L. Gazzolo and K. Mac6 Laboratoire d'Immuno- Virologie Mol6culaire et Cellulaire, UMR 30 CNRS/UCBL, Facuit6 de Mddecine Alexis Carrel, 69372 Lyon Cedex 08 (France)

Summ~ry. Viral infection of immunocompetent cells always leads to disordered regulation of the immune system. Thus, infection by HIV1 (human immunodeficiency virus, type 1) of mononuclear phagocytes and lymphocytes is linked to the induction of the acquired immunodeficiency syndrome (AIDS). HIVI replication in mononuclear phagocytes appears to be dependent on both the stage of maturation and on differentiation of mononuclear phagocytes. Because of the heterogeneity of the mononuclear phagocyte system, the U937 cell line provides a convenient model for studying the regulation of HIV1 replication in mononuclear phagocytes and the involvement of these cells in the immunopathogenesis of HIVI. We have shown that endogenous interferon alpha (IFNa) restricted viral replication in these promonocytic cells, probably by acting on proviral transcription and by interfering with transcriptional factors involved in the transactivation of the LTR (long terminal repeat) of HIV1. Indeed, addition of a monoclonal antibody to IFN~ to U937 cells cotransfected wJui a LTR n~v linked to the bacterial chloramphenicol ac~y~ transferase (CAT) gene, together with a tat expression vector, leads to an increase in CAT activity. Conversely, the addition of IFNct to cells cotransfected with the same vectors is followed by a decrease in CAT activity. Finally, recent experiments indicate that chronically HIV-l-infected U937 cells are more differentiated than uninfected U937 ceils, suggesting that viral gene expression is able to trigger the maturation process of the promonocytic cells towards a stage where viral transcription may escape IFNa. These results suggest that the first replicative cycle of HIVI in monocytes/macrophages could be a unique target for therapeutic strategies based on the use of IFN=. .

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KEY.wogos: SIDA, HIVI, Interferon, Transcription, Promonocyte; U937 cell, Replication, Transactivation.

Introduction. Viral tropism for unique target cells most often governs the development of specific diseases following the infectious process. Thus, viral infection of immunocompetent cells always leads to a disordered regulation of the immune system (McChesney. and Oldstone, 1987). For example, mononuclear phagocytes, when infected by runnnant lentiviruses, initiate diseases with inflammatory and/or degenerative changes in the immune and central nervous systems (Narayan et al., 1985). One special feature of lentivirus replication is its dependence on the stages of both maturation and differen-

260

L. GAZZOLO

AND

K. MACE

tiation of the infected cells. Indeed, whereas viral transcription is null or very low in bone marrow or blood monocytes, viral gene expression increases several thousandfold when monocytes mature into tissue macrophages (Gendelman et al., 1986). Human immunodeficiency virus type 1 (HIV 1) shares many biological, biochemical and molecular properties with lentiviruses (Gonda et al., 1985; Chiu et aL, 1985). Monocytes and lymphocytes represent the major target cells of HIVI, and both types of cells may be latently infected by the vhus (Rosenberg and Fauci, 1989). Viral expression, when triggered by inductive signals, causes the death of lymphocytes, whereas monocytes and macrophages are relatively resistant to the cytopathic effects of the virus, ir,dicating that these cells may harbour the virus for a long period of time before clinical symptoms become apparent (Roy and Wainberg, 1988). The~,efore, we were interested in investigating, at the cellular and molecular levels, factors iatervening in the maintenance of latent infection of monocytes/macrophages. Because of the heterogeneity of the mononuclear phagocyte system and the difficulty in obtaining pure cell populations, we chose to study HIVI replication in U937 cell line. These human promonocytic cells, derived from a patient with generalized histiocytic lymphoma, differentiate into mature macrophages in the presence of phorbol-I 2-myristate-I 3-acetate (PMA) (Sundstrom and Nilsson, 1976). They become adherent and express the phenotypic properties of macrophages, including the expression of Fc3" and CD3 receptors (Minta et ¢L, 1985). Materials and methods. Cellline and cell culture. - - U937 is a promonocytic leukaemia cell line established from a human histiocytic lymphoma (Sundstrom and Nilsson, 1976). Cells were grown in RPMI-1640 supplemented with 10 °70 foetal calf serum (FCS), glutamine (2 mM) and antibiotics (penicillin 100 IU/ml, streptomycin 50 Izg/ml). Infection and anti-IFN treatment. ~ U937 cells were incubated with a cell-free supernatant (2 x 105 cpm/ml) from HIVl-infected CEM cells, as previously described (Mac6 et al., 1989). Monoclonal antibody (mAb) to human IFNa, kindly providc,el h w I'~r T ~ a l l r ~ n t

uz~a© o e i c h a r l / A | l i ~ t J a r l 1 /qIfl/~X a t ¢ h ~ t | r n a ~4" ~r~'ar-t;e~r~ , a n t i r n o ; n t ~ r ~ ,

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thereafter. Plasmids. ~ LTRHIv-CAT and the pSVz-CAT constructs: HIV1 and simian virus 40 (SV40) promoters, respectively, linked to the chloramphenicol r,cetyl transferase (CAT) gene. These plasmids were kindly provided by Dr Virelizier and Dr Sergeant, respectively. The tat/rev expression vector (pSVz-TATA) was kindly provided by Dr Emerman. Transfection and C A T assay. ~ A total of 107 cells were transfected using a DEAE (diethylaminoethanol) dextran method. Briefly, cells were incubated for 5 min at 37°C, then 30 min at room temperature in 1.2 ml of the transfection solution

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ed once in TBS, resuspended in RPMI containing 10 070 foetal calf serum and incubated for 43 h. When needed, incubation was performed in the presence of mAb to IFNGt(diluted 1/200) or in the presence of IFNQt (250 U/ml). Cells were harvested

CAT DEAE IFN LTR

= chloramphenicol acetyl transferase. = diethylaminoethanol. = interferon. = long terminal repeat.

mAb PMA SV TBS

= = = =

monoclonal antibody. phorbol-12-myristate-13-acetate. simian virus. Tris buffer solution.

HIV1 R E P L I C A T I O N I N U937 P R O M O N O C Y T E S

261

and assayed f~r CAT activity. CAT assay reaction mixtures contained 80 ~tl of lysate, 0.15 i~Ci of 14C-chloramphenicol (CEA, Gif-sur-Yvette, France) and 20 ~tl of 0.4 mM acetyl coenzyme A (Bohreinger, Mannheim, FRG). Reaction mixtures were incubated for 2 h at 37°C (Gorman et al., 1982). Results and discussion.

H I V I replication in U937 cells is restricted by IFN¢.

We first compared the replication of HIV1 in U937 cells and in H9 cells, a CD4 T-lymphoblastoid cell line. Most of the T lymphoblasts were positive for the viral core antigen p24, 7 to 10 days after infection. In contrast, a delay of about 40 days after viral infection occurred before the same percentage of U937 cells was positive for p24 (fig. 1). When U937 cells were treated with an mAb to IFN¢, and infected with HIVI, the number of p24-positive cells was found to increase in the treated HIVl-infected U937 cells much earlier than in the untreated HIVl-infected cells (fig. 1). This experiment and other experiments (Mac~ et al., 1989) indicate that endogenous IFN= restricts viral replication in promonocytic cells. Thus, the HIVI/U937 system is a convenient model for studying the regulation of HIV 1 replication in the mononuclear phagocytic cells and the involvement of these cells in the immunopathogenesis of HIVI. 100" 80 ca

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Time after infection (days)

FIG. 1. -- Effect o f mAb to human IFN= on p24 expression by U937 cells infected with HVI1. Expression of viral p24 antigen was determined by indire~'timmunofluorescence, using a mouse mAb specific to the p24 core protein, as previously described (Mac~ et al., 1989).

Inhibition by IFN~ o f L T R - H I V transactivation.

Antibody to IFN~ has been reported to increase the yield of HIVI in peripheral blood mononuclear cells from AIDS patients (Barr6-Sinoussi et al., 1983). One hypothesis is that this neutralizing antibody favours HIV1 viral transcription, gene expression and viral release from monocytic cells. Studies with routine retroviruses indicate that IFN¢ inhibits early events in viral replication such as transcription or translation of the viral genome during acute infection, and interferes with virus assembly and release in chronically infected cells (Pitha et al., 1979). We therefore

L. G A Z Z O L O A N D K. MACI~

262

studied the effect of IFN~ antibody and that of IFN~ on the transactivating effects exerted by the tat-gene product on the expression of a chimeric gene. For these experiments, the LTR HIV was linked to the bacterial CAT gene. In vitro, this construct directs high levels of CAT activity only in cells producing the protein encoded by the tat gene. The tat protein is a powerful transactivator of HIV gene expression with one or several levels of control (Cullen, 1986; Rosen et al., 1986). Table I shows the results of an experiment in which U937 cells were transfected with the LTR CAT construct alone or together with a tat/rev expression vector under the control of the SV40 promoter. A 40-fold increase in CAT activity was obtained in the presence of the tat-gene product, and the addition of an IFN0t antibody led to a two-fold increase when compared to the untreated cultures. Likewise, the addition of IFNct to the cotransfected U937 cells was followed by a significant decrease (about 5-fold) in CAT activity. No effect was observed on the expression of the CAT gene under the control of the SV40 promoter (pSV2-CAT) in U937 cells treated by IFN~ or by an IFN~t antibody (table I). Taken together, these results indicate that IFN~ might act at the proviral transcription level by interfering with the transactivating effect of the tat protein. TABLE I. - - Transaetivation o f L T R m v - C A T by the tat/rev proteins: effect o f m A b to IFN~ or IFN~ in U937 cells.

Transfec~ed plasmids

Additive

Percent acetylated chloramphenicol

LTRmv-CAT LTRmv-CAT + tat/rev LTRmv-CAT + tat/rev pSV2-CAT pSV2-CAT LTRmv-CAT

none none anti-IFN~ none anti-IFNct none none IFNQt none IFN~

1.0 40.0 76.0 8.0 5.0 0.3 79.5 17.0 4.3 4.5

I .TR ...... CAT

4- t a t ~ r o y

E'i'i~iv-CA'i + tat?rev pSVz-CAT pSV2-CAT TM

Percentage acetylated chloramphenicol = cpm o f acetylated chloramphen;eol/total cpm.

Differentiation stage o f U937 cells chronically infected with HIVI. The above experiments suggest that IFN~t inhibits the transactivating effects of the tat protein. However, the observation that the viral replication in U937 cells was delayed but not totally suppressed suggests a complex interaction of negative and inductive factors on viral activation. In vitro, among the variety of inductive signals triggering HIV1 replication in latently infected U937 cells, are cytokines (Folks et al., 1987) and differentiating agents such as PMA (Poli et al., 1989). Interestingly, when chronically HIV-infected U937 cells were treated with PMA, the majority of HIVl-infected cells were attached to the surface of the culture dish 5 rain later, at which time uninfected U937 cells treated with PMA were still not adherent (fig. 2). In these uninfected cultures, 20 rain after addition of the drug, the number of adherent cells is still 5-fold less than that in HIV U937 cells. This experiment suggests that the U937 cells chronically infected by HIVI are more differentiated than the uninfected cells. One plausible explanation is that the viral regulatory proteins, and especially

H I V I R E P L I C A TION I N U937 P R O M O N O C Y T E S

263

the tat protein, are able to drive the differentiation of U937 cells towards a stage where viral transcription may escape negative cellular factors, such as IFNat. Experiments are now in progress to test this hypothesis. 120

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U937 U937/HIV

80

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60 40 2O

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0 5 min.

20 min.

Time after addition of PMA i

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Effect o f PMA on the adherence capacity o f U937 and U937/HIV cells.

Cells (106/ml) were incubated in RPMI supplemented with 10 °70 foetal calf serum and 50 ng/ml of PMA, for 5 or 20 min, at 37°C. Adherent cells were washed once in PBS, coloured by Giemsa for 5 min then washed several times in PBS. Adherent cells present in 4 different optical fields were counted using a Nikon microscope at a magnification of x I00.

An early event in the differentiation process is the induction, at a high level, of the nuclear protein encoded by the c-fos gene in differentiated macrophages (Miiller, 1986). Additionally, the Fos protein, which complexes with the cellular-binding protein AP-i (Sassone-Corci et aL, i988), seems to be responsible for visna virus activation, with the viral LTR containing AP-1 sites (Hess et al., 1989). Preliminary experiments (data not shown) indicate that cotransfection of human c-fos and LTR HIV CAT constructs results in a higher level of CAT expression than with LTR HIV CAT alone. Nevertheless, because the LTR HIV does not contain AP-1 sites, the Fos protein might act indirectly, perhaps by induction of cellular transcriptional factors such as Spl and NFk-B involved in LTR HIV activation (Jones et al., 1986; Nabel and Baltimore, 1987). Since IFNQt downregulates c-fos expression (Einat et al., 1985), it is tempting to speculate that this interplay (fig. 3) is pivotal in controlling the transactivating effect of the viral tat protein and the release of viral particles. Tat protein

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IFN a l p h a