JOURNAL OF VIROLOGY, Sept. 1997, p. 6495–6500 0022-538X/97/$04.0010 Copyright © 1997, American Society for Microbiology
Vol. 71, No. 9
Modulation of RANTES Production by Human Cytomegalovirus Infection of Fibroblasts SUSAN MICHELSON,1* PAOLA DAL MONTE,2 DONATO ZIPETO,1 BAHRAM BODAGHI,1 LYSIANE LAURENT,1 ESTELLE OBERLIN,1 FERNANDO ARENZANA-SEISDEDOS,1 JEAN-LOUIS VIRELIZIER,1 AND MARIA PAOLA LANDINI2 Unite´ d’Immunologie Virale, Institut Pasteur, Paris, France,1 and Department of Clinical and Experimental Medicine, Division of Microbiology, University of Bologna, Bologna, Italy2 Received 3 March 1997/Accepted 2 June 1997
Chemokines play a major role in inflammatory responses and affect hematopoiesis both negatively and positively. We show that fresh isolates and laboratory strains (Towne and Ad-169) of human cytomegalovirus (HCMV) induce production of the CC chemokine RANTES in fibroblasts. Induction of extracellular RANTES production occurred as early as 8 h after infection, peaked around 24 h after infection, and was almost undetectable by 48 and 72 h. Upregulation occurred in the absence of viral DNA synthesis, suggesting that it was due to immediate-early–early HCMV gene expression. CMV infection stimulated RANTES transcription, since reverse transcription-PCR detected a sharp increase in RANTES RNA which persisted even when extracellular RANTES was no longer detected. Induction of RANTES in fibroblasts was not due to prior induction of tumor necrosis factor alpha or interleukin 1b. Down-regulation required an active viral genome. Decrease of RANTES in culture supernatants may be associated with the appearance of the HCMV CC chemokine receptor US28, since we show that this gene is transcribed as early as 8 h after infection. Modulation of CC chemokine production early during CMV infection might have a regulatory effect on viral replication, as well as affect immune surveillance. nence of this model to human CMV-caused pneumonia (13). In vivo studies suggest that CMV infection can modify the level of cytokines with chemoattracting properties known as chemokines. Monti et al. (23) studied the level of RANTES production by cells recovered by bronchoalveolar lavage from lung transplant patients with CMV pneumonitis and showed that cells from infected patients secreted greater amounts of RANTES than did cells recovered either from patients undergoing acute rejection or from control subjects. A study by Bernasconi et al. (5) showed that AIDS patients with CMV encephalitis have markedly higher concentrations of MCP-1 but not other chemokines in their spinal fluid than do human immunodeficiency virus-seropositive persons who are asymptomatic or AIDS patients with a number of other opportunistic infections of the central nervous system. In light of these in vivo studies, we thought it important to investigate the modulation of CC chemokine production upon in vitro CMV infection of fibroblasts that have been shown to be targets for CMV infection in vivo (33, 35). We show that RANTES mRNA and protein are induced early during CMV infection of fibroblasts. In addition, extracellular RANTES accumulation, but not transcription, is down-regulated in fibroblasts late during CMV infection. This is accompanied by accumulation of RANTES in the cytoplasm of infected cells. (Part of this work was presented at the 6th International CMV Workshop, 5 to 9 March 1997, Perdido Beach, Ala.)
Cytomegalovirus (CMV) is a major cause of morbidity and mortality in immunocompromised individuals and of congenital malformations if acquired in utero. The presence of CMV-infected cells at a site is frequently accompanied by lymphocytic and monocytic infiltrations and inflammation. Accumulation of immune cells within an organ involved in an inflammatory reaction is caused by the migration of circulating blood cells to the site of the pathological process. Migration of circulating cells is induced by cytokines with chemoattracting properties, referred to as chemokines. CMV organ involvement is frequently accompanied by infiltration of monocytes and lymphocytes and the presence of CMV-infected cells with normal morphology (8, 18, 24, 39). Chemokines are chemoattractants for neutrophils, monocytes, lymphocytes, and bone marrow progenitors (1), as well as other cell types (reviewed in references 3, 25, and 32). The family of chemokines comprises four subfamilies defined by the distribution of cysteine residues in the N terminus of these factors: the CXC, CC, C, and CX3C (4) subfamilies. While it has been shown that CMV encodes a functional receptor for one subfamily of chemokines (12, 19, 27), no in vitro study has been done on the effect of CMV infection on chemokine production by various cell types. Such studies could provide important information, since two in vivo studies have suggested that CMV infection can indeed modify chemokine production, although the cells implicated in these modifications have not been defined (5, 23). Certain CMV diseases are of an immunopathological nature rather than due solely to virus replication. This has been clearly illustrated in the murine CMV model of interstitial pneumonia by Grundy et al. (14), who subsequently discussed the perti-
MATERIALS AND METHODS Cells. Foreskin fibroblasts (FSF; a gift of C. Paya, Mayo Foundation, Rochester, Minn.) were maintained in Dulbecco’s modified Eagle’s medium–10% decomplemented fetal calf serum (FCS) and used between passages 15 and 33. Various continuous human cell lines were also studied. Astrocytoma cells (U373MG), known to be permissive for CMV replication (10), were originally obtained from R. LaFemina (Merck Sharp & Dohme, West Point Pa.) and grown in Dulbecco’s modified Eagle’s medium–5% FCS. Myeloprogenitor cells (TF-1, HL-60, and U937) were grown in RPMI medium–10% FCS. Lymphoid cell lines (Jurkat and a subclone, JJhan) were maintained in RPMI medium–5% FCS. All cells were periodically tested for mycoplasma and found to be negative.
* Corresponding author. Mailing address: Unite´ d’Immunologie Virale, Institut Pasteur, 28 rue du Dr. Roux, 75724 Paris Ce´dex 15, France. Phone: (33 1) 45 68 82 64. Fax: (33 1) 45 68 89 41. E-mail:
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Viruses. Two laboratory strains of CMV were used: Towne and Ad-169. Fibroblasts were infected at 1 PFU/cell. Twelve clinical isolates were also studied (for their origins, see Table 2). These strains were isolated on human diploid fibroblasts by techniques routinely used in diagnostic laboratories. Herpes simplex virus (HSV) strain KOS was originally provided by F. Rapp (Hershey Medical Center, Hershey, Pa.). All virus stocks were grown and their titers were determined on human diploid fibroblasts, and they were negative for mycoplasma. Generation of supernatants and measurement of CC chemokines. Laboratory strains of CMV were adsorbed to cells at a multiplicity of infection of 1 for 1 h at 37°C. Medium was changed on mock-infected cells. Cells were then washed once with phosphate-buffered saline and refed growth medium. In some experiments, neutralizing anti-tumor necrosis factor (TNF), anti-interleukin 1 (IL-1), or control antibodies (generously provided by Roussel UCLAF, Paris, France) were added (5 mg/ml) to culture supernatants along with virus and maintained in the medium throughout the study period. To inhibit CMV DNA synthesis, infected cells were treated continuously with phosphonoacetic acid (PAA; 200 g/ml; generous gift from Abbott Laboratories) (16) starting immediately after virus adsorption. Uninfected cells were treated in parallel. Fibroblasts were also infected with HSV at a multiplicity of infection of 1, and culture supernatants were sampled 2, 4, and 24 h postinfection (hpi), clarified as described below, and stored at 280°C. Supernatants were collected from CMV-infected cells at the times indicated in Results and in the tables. Upon sampling, supernatants were clarified by centrifugation at 800 3 g for 10 min at 4°C and kept frozen at 220 or 280°C until tested. RANTES was quantitated by using enzyme-linked immunosorbent assay (ELISA) kits from R&D Biotechnology (R&D Systems, Abingdon, United Kingdom) or Medigenix (Biosources) in accordance with the manufacturers’ instructions. Comparable results were obtained with kits from both sources. All results are expressed as picograms per milliliter of unconcentrated culture supernatant as calculated by linear regression from a curve of six standards run in each test. Isolation of clinical isolates. Clinical specimens (urine, saliva, polymorphonuclear leukocytes, liver biopsies) from subjects already known to excrete CMV or at high risk of being in an active phase of CMV infection were inoculated onto monolayers of FSF and left for 2 h at 37°C. The inoculum was then removed, and new medium was added to the cells. Incubation was carried out at 37°C in a CO2 incubator. Cytopathic effects (CPE) usually occurred after 7 to 14 days. When the CPE involved more than 20% of the cells, infected cells were split. After 3 to 10 passages, depending on when 100% CPE were obtained, the supernatants of infected cells were clarified by centrifugation (800 3 g, 30 min) and used to infect human lung fibroblasts to study RANTES induction. The titer was determined after freeze-thawing once. Reverse transcriptase (RT)-PCR analyses of RANTES transcription. RT-PCR was used to analyze transcription of RANTES and of US28 following infection of fibroblasts with the Ad-169 strain of CMV. RNA was extracted from cells by using RNeasy (Qiagen S.A., Courteboeuf, France) in accordance with the manufacturers’ instructions. Total extracted RNA was treated with RNase-free DNase, repurified, and quantitated spectrophotometrically. One microgram of RNA was retrotranscribed in PCR buffer (1.5 mM MgCl2) containing 100 pmol of random primers, 1 mM each deoxynucleoside triphosphate, and 200 U of Superscript reverse transcriptase (Gibco-BRL) by incubation at 25°C for 10 min and at 42°C for 60 min in a final volume of 20 ml. The resulting cDNAs were amplified by using 30 pmol of the following primers for RANTES: upstream primer, 59 CGG GAT CCA TGA AGG TCT CCG CGG CA-39; downstream primer, 59 CGG AAT TCC TAG CTC ATC TCC AAA GA-39. The following primers were used for US28: upstream primer, 59-ATG ACA CCG ACG ACG ACG ACG G-39; downstream primer, 59-GCT AGG GAG TTG TGA TCT AG-39. Amplification was performed with a Thermocycler 9600 (Perkin Elmer) at 94°C for 30 s, 57°C for 30 s, and 72°C for 1 min for a total of 30 cycles. Immunofluorescence assay for intracellular RANTES detection. For detection of intracellular RANTES, a goat anti-RANTES polyclonal antibody (R&D Systems) was used, followed by fluorescein isothocyanate-conjugated anti-goat immunoglobulin G serum.
RESULTS To determine whether CMV infection can modulate RANTES production during its replication in human fibroblasts, FSF were infected and supernatants from infected and mock-infected cells were collected from the end of the virus adsorption period to the times indicated in Fig. 1, which summarizes 10 experiments. The RANTES levels and the kinetics of RANTES appearance in supernatants of fibroblasts infected with the Towne and Ad-169 strains were the same (data not shown). The time of appearance of RANTES in the supernatant (within 8 hpi) corresponds to the immediate-early and early phases of the virus cycle. The amount of RANTES increased at 16 and 24 hpi. In most of the instances shown, there
J. VIROL.
FIG. 1. RANTES induction and down-regulation following CMV infection of fibroblasts. Fibroblasts were infected, and supernatants were collected at the times indicated under the graph. Results of 10 experiments are given. The curve corresponds to the median values for all experiments. Induction was observed as early as 8 hpi, peaked at 16 to 24 hpi, and decreased notably at 48 hpi.
was a significant reduction in the amount of RANTES found at later times after infection. The passage level of the fibroblasts used as the substrate for infection did not play a role in the amount of RANTES induced or the kinetics of production (data not shown). It has been reported that TNF-a and IL-1 (28, 30, 34) can induce production of RANTES in fibroblasts. By using the same cells, we showed previously that CMV infection induced neither TNF-a nor IL-1 (21). However, to determine whether induction of RANTES during CMV infection was mediated in part by these cytokines, we infected fibroblasts with CMV and added 5 mg of either neutralizing anti-TNF-a, anti-IL1, or control antibodies per ml at the end of the 30-min adsorption period. Supernatants were collected at 8, 16, and 48 h after infection. No difference was seen in either the kinetics or the level of RANTES induced in the presence of these antibodies (averages of three experiments at 16 hpi: 288.0 pg/ml with anti-TNF-a antibody, 413.0 pg/ml with anti-IL-1 antibody, 307.2 pg/ml with control antibody, and 267.5 pg/ml in untreated, infected cell supernatants). Supernatants of uninfected cells incubated with any of the antibodies contained no RANTES, and samples not treated contained RANTES at 19.5 pg/ml. To exclude the possibility that the observed induction of RANTES was a general phenomenon linked to herpesvirus infection of fibroblasts, we repeated the experiments by infecting fibroblasts with HSV. Infection with HSV did not induce the appearance of RANTES at any of the times studied, despite clear development of CPE in approximately 95% of the culture at 24 h after infection (data not shown). To examine more precisely the presence of RANTES in the supernatants of fibroblasts as a function of time after infection, supernatants were collected at different intervals after infection from the same wells, with refeeding of cells at each time point. The results shown in Fig. 2 confirm that peak production occurred by 24 h and show that decreasing amounts of RANTES were detected in the supernatant of infected cells after that time. To exclude the possibility that RANTES induction is a phenomenon linked to infection by laboratory strains of CMV, 12 clinical isolates (between in vitro passages 3 and 10) were tested for the ability to induce this chemokine (Table 1). Isolates were divided into three groups. Group 1 isolates had the highest titers, produced maximum CPE, induced early appearance of RANTES in supernatants, and later caused a decrease. These kinetics were superimposable on those observed with laboratory strains. Group 2 consisted of isolates with moderate
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FIG. 2. Down-regulation of RANTES production in the absence of CMV DNA synthesis. Fibroblasts were mock infected or infected with the Ad-169 strain of CMV (1 PFU/cell) and then treated with PAA (200 g/ml) at the end of the virus adsorption period or were left untreated. Supernatants were collected at the time intervals indicated, and the medium was changed in the remaining wells. RANTES production in supernatants was measured by ELISA as indicated in Materials and Methods. Light bars, untreated cells; dark bars, cells treated with PAA. These results are representative of four separate experiments.
titers which produced intermediate CPE. With these isolates, RANTES production was delayed and a total decrease was not seen at 48 h, probably because these isolates grew more slowly. We therefore prolonged the observation period to 72 h and found that in three cases (isolates Bon2, Man, and Rea), RANTES levels decreased. Group 3 isolates had the lowest titers, yielded little CPE, and showed no clear RANTES induction. The modulation of extracellular RANTES production induced by CMV thus appears to be biphasic: an initial, rapid induction followed by disappearance of RANTES from infected cell supernatants.
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The role of virus particles in this induction was investigated by using pelleted virus and virus-free supernatants. The results in Table 2 indicate that pelleted virus induced both phases of RANTES modulation in a manner identical to that of a wholevirus inoculum. When pelleted virus was inactivated by either heat or UV irradiation, rapid and marked RANTES induction was observed which persisted for the first 48 hpi and showed only a slight decrease at 72 hpi. Clarified supernatants gave rise to a delayed, much-reduced RANTES induction. Disappearance of extracellular RANTES was not complete by the latest observation time. When these supernatants were filtered through a 30-kDa exclusion membrane, the induction observed at 24 h was reduced and inapparent at later times. Although supernatants had been ultracentrifuged twice, particles capable of inducing low-level immediate-early protein expression were still present, as determined by immunofluorescence (data not shown). To determine if the RANTES decrease at later times is a true late event, we specifically blocked CMV DNA synthesis by addition of PAA (16) at the end of the adsorption period. Figure 2 shows that neither the induction of RANTES after infection nor its subsequent decrease was affected by the addition of PAA. To see if RANTES was being destroyed by proteases in infected cell supernatants, we incubated a given amount of recombinant RANTES in supernatants from uninfected cells and from cells infected for 48 h which were negative for RANTES by ELISA. Incubation was carried out overnight at 37°C. Controls consisted of the same amount of RANTES incubated at 37°C overnight in growth medium alone and the same amount of RANTES diluted in growth medium and frozen at 220°C overnight. There was no notable loss of RANTES upon incubation at 37°C in infected cell supernatant (1,282.6 pg/ml) compared to that incubated in uninfected cell supernatant (1,390.9 pg/ml) or in growth medium alone (1,678.4 pg/ml); the amount of RANTES in growth medium kept at 220°C was 1,536.7 pg/ml. To further explore the mechanism(s) involved in the reduction of the amount of RANTES in the supernatants of CMVinfected fibroblasts, indirect immunofluorescence was performed with a polyclonal antiserum to RANTES (R&D Biotechnology). As shown in Fig. 3, in uninfected cells and
TABLE 1. Induction of RANTES by infection of fibroblasts with fresh CMV isolates Isolatea
Sourceb
RANTES production (pg/ml)
Titerc
CPEd
Uninfected cells
0–24 hpi
24–48 hpi
48–72 hpi
Lon Mas Bia Gir2
Urine (N) Saliva (N) Blood (I) Urine (RTR)
1.6 3 104 1 3 104 1.2 3 104 1.6 3 104
0 0 0 0
15.8 368.2 59.9 53.6
0 0 0 0
0 0 0 0
Bon2 Man Rea Car
Saliva (I) Urine (PW) Urine (N) Liver biopsy (F)
3.2 3 103 4.3 3 103 4.5 3 103 2.1 3 103
0 0 3.1 6.8
0 9.1 115.1 3.1
11.8 43.8 360.0 13.2
0 20.0 237.7 14.4
11 11 11 11
Pom2 Gir Bon Pom
Urine Urine Saliva Urine
2.4 3 103 1.1 3 103 0.8 3 103 0.7 3 103
0 10.6 0 7.2
0 9.8 4.9 5.7
0 7.6 5.3 21.9
11.8 0 2.7 23.1
1/2 1/2 1/2 1/2
a
(N) (RTR) (I) (N)
Isolates whose names end in the number 2 were passaged another time to try to increase their infectivity. N, newborn; I, infant; PW, pregnant woman; F, fetus; RTR, renal transplant recipient. Number of infectious particles per milliliter determined on stocks used for infection. d Estimate of CPE in cultures at the end of the collection period. 111, 70 to 90%; 11, 40 to 70%; 1/2, ,40%. b c
111 111 111 111
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TABLE 2. Modulation of RANTES production by pelleted CMV and clarified supernatants Prepn or treatment
Inoculum Pelleted virus UV inactivatedb Heat treatedc Supernatant Filtered supernatant a b c
RANTES concn (pg/ml) in supernatant at time indicated after contact with cells 0–8 h
0–24 h
24–48 h
1,509 6 80 2,093 6 118 3,261 6 194 3,584 6 193 0 NDa
3,649 6 109 3,852 6 125 4,189 6 206 4,292 6 147 1,142 6 81 630 6 100
0 0 3,730 6 226 4,029 6 204 1,291 6 142 75 6 55
ND, not determined. UV irradiation resulted in a 5-log decrease of the original titer. Heated at 56°C for 30 min.
laboratory strains of CMV, since several fresh clinical isolates also induced RANTES. It did not appear to be an indirect induction due to prior induction of TNF-a or IL-1, which is known to induce RANTES production in fibroblasts (28, 30, 34). Antibodies which specifically antagonize these cytokines had no effect on the level or kinetics of RANTES induction. CMV infection of fibroblasts has been shown to induce production of transforming growth factor beta 1 (21) and basic fibroblast growth factor (2) and to upregulate constitutive IL-6 production (21). We do not know if these cytokines or others may influence RANTES production in fibroblasts. The second phase of RANTES modulation (decreased detection of extracellular RANTES) could be due to one or more factors. It could be a lack of translation of specific transcripts,
cells infected for 24 h, there was no RANTES-specific fluorescence, whereas at 48 hpi, RANTES was detected intracellularly when supernatants were almost negative for RANTES by ELISA. Fluorescence at 120 hpi was the same as at 48 hpi (data not shown). A CMV homolog (US28) of CC chemokine receptors has been shown to bind RANTES (19). It is therefore conceivable that US28 receptor expression could play a role in the down-regulation of RANTES in infected cell supernatants if US28 is expressed earlier than originally reported (38). We therefore investigated the kinetics of appearance of both RANTES and US28 in infected fibroblasts. Cells were infected and then incubated for a total of 8, 24, and 48 h; RNA was extracted, treated with DNase, and retrotranscribed with random primers; and cDNA was amplified by using US28- and RANTES-specific primers. The results in Fig. 4 show that transcription of both RANTES and US28 RNA could be detected as early as 8 hpi and persisted throughout the study period. Uninfected cells were negative for both transcripts. We also tested for RANTES induction following infection of other cell types. None of the following cell types tested produced RANTES following CMV infection: the promyelocytic cell line TF-1, U937 cells, the lymphocytic cell lines Jurkat and JJhan, and U373MG astrocytoma cells (data not shown). DISCUSSION Infection of human fibroblasts with CMV resulted in rapid induction of RANTES, starting at 8 hpi and reaching a peak at 24 h as judged by its presence in culture supernatants and by the detection of specific transcripts in infected cells. Although RANTES transcription persisted late in the viral replicative cycle in fibroblasts, the presence of RANTES in the culture supernatants decreased. The early appearance of RANTES in the supernatants, as well as its subsequent decrease, occurred in the presence of PAA, which suggests that both events are independent of viral DNA synthesis. Thus, modulation of extracellular RANTES production seems to be biphasic: early transcription-translation with excretion lasting at least 24 h, followed by continued transcription in the absence of excretion but with intracytoplasmic accumulation. The first phase of RANTES modulation (induction) could be due to incoming viral proteins and/or immediate-early gene expression, since heat- and UV-inactivated viral particles induced similar levels of RANTES excretion and clarified supernatant did not. Incoming CMV-associated proteins have been shown to modify host cell protein synthesis and phosphorylation in the absence of any viral genome expression (6, 22). RANTES induction does not seem to be an artifact due to
FIG. 3. Immunofluorescent detection of RANTES at various times postinfection in CMV-infected cells. Cytoplasmic immunofluorescence was detected with a goat anti-RANTES polyclonal antibody in infected fibroblasts at 24 (A) and 48 (B) hpi with 1 PFU of strain Towne CMV per cell and in uninfected cells at 24 (C) and 48 (D) h after the beginning of the experiment. The amounts of RANTES (in picograms per milliliter) in the corresponding supernatants were as follows: uninfected cells, 0 at both times; infected cells at 24 h, 671.6; infected cells at 48 h, 90.7.
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The question of the pertinence of CC chemokine induction to CMV replication arises. In this regard, it has been shown that CMV open reading frame US28 encodes a promiscuous, calcium-mobilizing receptor for the CC chemokine RANTES, MIP-1, and MCP-1 (12, 19, 37). The chemokine selectivity that is a characteristic of the US28 receptor suggests a role for the CC chemokines in the pathogenesis of CMV disease by transmembrane signaling via the US28 product. This receptor is apparently transcribed early and thus could play a role in cellular activation. It may be incorporated into the virion envelope during maturation, as was recently shown for another CMV CCR receptor homolog, UL33 (20). The receptor(s) might then be incorporated into the cell membrane during fusion of the viral envelope with the plasma membrane upon viral entry (7, 36). RANTES adsorption to cellular and/or CMV-encoded receptors might then lead to activation of infected cells as soon as RANTES is induced. Experiments are in progress to determine whether US28 is present in the viral envelope. The early induction of CC chemokines might thus be important for the regulation of CMV replication. Induction of CC chemokine production by CMV could recruit lymphocytes and monocytes to sites of infection, thus participating in pathogenesis (CMV-induced lesions) and the control of infection by immunocompetent cells during primary infection, as well as playing a role through immunosurveillance in the control of reactivation from latency.
FIG. 4. Detection of RANTES, US28, and actin transcripts in CMV-infected fibroblasts by RT-PCR. Fibroblasts were infected and incubated for 8, 24, or 48 h or left uninfected. RNA was prepared, retrotranscribed, and amplified as described in Materials and Methods. RANTES transcripts were undetectable in uninfected cells. CMV infection induced RANTES transcripts by 8 hpi. Transcripts were still detected at 48 hpi, when levels of extracellular RANTES had returned to that of uninfected cells. RANTES concentrations (in picograms per milliliter of supernatant) were as follows: uninfected cells cultured for 48 h, 5 0; infected cells at 8 hpi, 276.9; at 24 hpi, 522.7; at 48 hpi, 34.6. Amplification of beta actin RNA in both cases showed that similar amounts of RNA were studied at each time point. US28 transcripts were detected at 8 hpi, and transcription continued up to the end of the study period (48 hpi). To control for the presence of DNA in RNA preparations, both sets of primers were used for amplification of nonretrotranscribed RNA (RT2). These results are representative of three separate experiments. Lane M contained molecular size markers. Lane NC contained only the PCR mix without RNA.
This work was supported by the Agence Nationale de Recherche sur le Syndrome d’Immunode´ficience Acquise and by the Biomed 2 European Concerted Action projects Infections with HCMV in the Immunocompromised Host and ROCIO II. D.Z. is a beneficiary of a Training and Mobility grant (ERBFMBICT961426) from the European Commission. B.B. is a beneficiary of a scholarship from Le Fonds d’Etudes de l’Assistance Publique, Ho ˆpitaux de Paris. B.B. was the beneficiary of grants from the Fondation Berthe Fouassier, the Fondation pour la Recherche Me´dicale, and Fonds d’Etudes et de la Recherche du Corps Me´dical des Ho ˆpitaux de Paris.
a lack of transport from the cytoplasm to the extracellular space, or the adsorption of RANTES to its cellular receptors (CCR1, CCR3, and CCR5) or to CMV homolog of CCRs (27) and its subsequent internalization, as shown for other chemokines (11, 26, 29, 31). Retention of RANTES in the cytoplasm seems the most likely possibility, since we detected RANTES protein by immunofluorescence in the cytoplasm of infected cells at late, but not early, times after infection. Sequestering in the cytoplasm could be related to the expression of CMV genes known to interfere with intracellular transport, as shown for HLA class I molecules (15, 17, 38). The presence of RANTES in the cytoplasm could also be due to internalization of RANTES following its excretion and receptor binding. However, preliminary results indicate that the fibroblasts we used do not express RANTES receptors CCR1 and CCR5, even after infection. CMV also encodes a functional RANTES receptor (US28) (12) which could be responsible for both internalization and sequestration of RANTES in the cytoplasm. This hypothesis can be envisaged because we have shown that US28 is effectively transcribed early after infection, contrary to a previous report (37). It has recently been reported that the product of another CMV CCR receptor homolog, UL33 (9), is transcribed within 4 h after infection and localizes to cytoplasmic inclusions late in the viral cycle (20). US28 might similarly localize to the intracytoplasmic inclusion body. We are currently investigating this possibility.
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