Cell, Vol. 85, 1135–1148, June 28, 1996, Copyright 1996 by Cell Press The ␤-Chemokine Receptors CCR3 and CCR5 Facilitate Infection by Primary HIV-1 Isolates

Hyeryun Choe,*# Michael Farzan,*# Ying Sun,* HIV-1 infects T lymphocytes, monocytes/macro- Nancy Sullivan,*† Barrett Rollins,§ Paul D. Ponath,‡ phages, dendritic cells and, in the central nervous sys- Lijun Wu,‡ Charles R. Mackay,‡ Gregory LaRosa,‡ tem, microglia (Gartner et al., 1986; Koenig et al., 1986; ‡ Walter Newman, Norma Gerard,k Craig Gerard,k Pope et al., 1994; Weissman et al., 1995). All of these and Joseph Sodroski*† cells express the CD4 glycoprotein, which serves as *Division of Human Retrovirology the receptor for HIV-1 and HIV-2 (Dalgleish et al., 1984; Dana-Farber Cancer Institute Klatzmann et al., 1984; Maddon et al., 1986). Efficient Department of Pathology entry of HIV-1 into target cells is dependent upon binding Harvard Medical School of the viral exterior envelope glycoprotein, gp120, to Boston, Massachusetts 02115 the CD4-amino-terminal domain (McDougal et al., 1986; † Department of Cancer Biology Helseth et al., 1990). After virus binding, the HIV-1 enve- Harvard School of Public Health lope glycoproteins mediate the fusion of viral and host Boston, Massachusetts 02115 cell membranes to completethe entry process (Kowalski ‡ LeukoSite, Inc. et al., 1987; Stein et al., 1987; Helseth et al., 1990). 215 First Street Membrane fusion directed by HIV-1 envelope glycopro- Cambridge, Massachusetts 02142 teins expressed on the infected cell surface leads to § Department of Medicine fusion with uninfected CD4-positive cells, resulting in Dana-Farber Cancer Institute syncytia (Lifson et al., 1986; Sodroski et al., 1986). Harvard Medical School Host cell factors in addition to CD4 have been sug- Boston, Massachusetts 02115 gested to determine the efficiency of HIV-1 envelope glycoprotein-mediated membrane fusion. Some human Perlmutter Laboratory k and animal cells were shown to be resistant to HIV-1 Children’s Hospital infection and syncytium formation even when human Department of Medicine CD4 was expressed on the cell surface (Maddon et al., Department of Pediatrics 1986; Ashorn et al., 1990; Chesebro et al., 1990; Beth Israel Hospital McKnight et al., 1994). Experiments with somatic cell Harvard Medical School hybrids suggested the possibility that a positive factor Boston, Massachusetts 02115 expressed in cells susceptible to syncytium formation could complement the block to fusion in resistant cell types (Clapham et al., 1991; Dragic et al., 1992; Broder et Summary al., 1993). HIV-1 variants exhibiting distinct differences in the ability to fuse with and to enter particular subsets We examined the ability of chemokine receptors and of CD4-positive cells have been identified (Broder and related G protein–coupled receptors to facilitate infec- Berger, 1995). All primary, clinical HIV-1 isolates, defined tion by primary, clinical HIV-1 isolates. CCR5, when as viruses that have not been passaged on immortalized expressed along with CD4, the HIV-1 receptor, allowed cell lines, replicate in primary monocytes/macrophages cell lines resistant to most primary HIV-1 isolates to and in primary T lymphocytes. Two groups of primary be infected. CCR3 facilitated infection by a more re- HIV-1 isolates have been defined, based on replication stricted subset of primary viruses, and binding of the rate in peripheral blood mononuclear cells (PBMC) and CCR3 ligand, eotaxin, inhibited infection by these iso- the ability to infect and induce the formation of syncytia lates. Utilization of CCR3 and CCR5 on the target cell in immortalized CD4-positive cell lines (A˚ sjo¨ et al., 1986; depended upon the sequence of the third variable (V3) Cheng-Mayer et al., 1988; Fenyo¨ et al., 1988; Tersmette region of the HIV-1 gp120 exterior envelope glycopro- et al., 1988). Most primary HIV-1 viruses that initiate tein. The ability of various members of the chemokine human infection and that persist throughout the course receptor family to support the early stages of HIV-1 of infection replicate to low levels in PBMC and do not replicate in immortalized T cell lines (A˚ sjo et al., 1986; infection helps to explain viral tropism and ␤-chemo- ¨ kine inhibition of primary HIV-1 isolates. Schuitemaker et al., 1991, 1992; Connor et al., 1993; Connor and Ho, 1994a, 1994b). These viruses are re- ferred to herein as macrophage-tropic primary isolates. In someHIV-1-infected individuals, viruses that replicate Introduction to higher levels in PBMC and that can infect and induce the formation of syncytia in immortalized CD4-positive Human immunodeficiency viruses type 1 and type 2 cell lines emerge late in the course of infection (A˚ sjo¨ et (HIV-1 and HIV-2) are the etiologic agents of acquired al., 1986; Schuitemaker et al., 1992; Connor et al., 1993; immunodeficiency syndrome (AIDS) in humans (Barre´ - Connor and Ho, 1994a, 1994b). These viruses will be Sinoussi et al., 1983; Gallo et al., 1984). AIDS results referred to herein as T cell line–tropic primary viruses. from the depletion of CD4-positive T lymphocytes in The T cell line–tropic primary viruses, by virtue of their HIV-infected individuals (Fauci et al., 1984). ability to replicate in some immortalized cell lines, serve as precursors to the laboratory-adapted isolates, which #The first two authors contributed equally to the work reported in have been extensively passaged on such cell lines. Lab- this paper. oratory adaptation results in a loss of the ability of HIV-1 Cell 1136

to replicate in primary monocyte/macrophage cultures The large family of G protein–coupled receptors re- (Chesebro et al., 1991; Schuitemaker et al., 1991; West- sponds to chemoattractants, neurotransmitters, pep- ervelt et al., 1992; Valentin et al., 1994). Thus, while all tide hormones, light, and odorants. Amino acid identity HIV-1 isolates replicate in primary T lymphocytes, three among receptors that bind functionally related ligands groups of virus variants can be defined based on the ranges from 20%–80% (Probst et al., 1992; Gerard and ability to replicate in primary monocyte/macrophages Gerard, 1994). Seven transmembrane receptors that or in immortalized T cell lines. The macrophage-tropic transduce their signals through heterotrimeric G pro- primary viruses cannot infect T cell lines, laboratory- teins are used by leukocytes to respond to chemokines adapted viruses cannot infect primary monocytes/mac- (Horuk, 1994). Chemokines are a family of structurally rophages, and T cell line–tropic primary viruses exhibit related peptides that recruit leukocytes to inflammatory dual-tropism for these cell types. lesions, induce release of granule contents from granu- Changes in the viral envelope glycoproteins, in partic- locytes, regulate integrin avidity, and in general exhibit ular in the third variable (V3) region of the gp120 exterior proinflammatory properties. The ␣ chemokines, or CXC envelope glycoprotein, determine tropism-related phe- chemokines, primarily act upon neutrophils, while the notypes (Cheng-Mayer et al., 1990; O’Brien et al., 1990; ␤ chemokines, or CC chemokines, generally act upon Hwang et al., 1991; Westervelt et al., 1991, 1992; Chese- monocytes, lymphocytes, basophils, and eosinophils bro et al., 1992; Willey et al., 1994). Amino acid changes (Baggiolini et al., 1994; Schall and Bacon, 1994). Thus, in the V3 region (Helseth et al., 1990; Freed et al., 1991; the CC chemokine receptors potentially exhibit a tissue Ivanoff et al., 1991; Bergeron et al., 1992; Grimaila et al., distribution consistent with the known tropism of HIV-1. 1992; Page et al., 1992; Travis et al., 1992) and the bind- While there are a number of closely related molecules ing of antibodies to this domain (Putney et al., 1986; in the CC chemokine receptor family, five of these have Goudsmit et al., 1988; Linsley et al., 1988; Rusche et al., been characterized in ligand binding assays. These are 1988; Skinner et al., 1988; Javaherian et al., 1989) have designated CCR1, CCR2A, CCR2B, CCR3, CCR4, and been shown to disrupt a virus entry process other than CCR5. (A uniform and simplified nomenclature for the CD4 binding. The target cell dependence of the pheno- ␤-chemokine receptors is currently under discussion. type resulting from V3 structural variation suggested We utilize CCR as the contraction for CC chemokine that the V3 region, which contains a surface-exposed, receptor. The subdesignations 1, 2A, 2B, 3, 4, and 5 are disulfide-linked loop (Leonard et al., 1990; Moore et al., those commonly used by investigators in the field.) Here 1994), might act in conjunction with target cell moieties we examine the potential role of these and other related to determine the efficiency of membrane fusion events. receptors in the infection of cells by primary HIV-1 exhib- Recently, an “orphan” G protein–coupled seven- iting macrophage-tropic and T cell line–tropic pheno- transmembrane-segment receptor, variously called types. HUMSTSR, LCR-1, or LESTR (Federsppiel et al., 1993; Jazin et al., 1993; Loetscher et al., 1994) has been shown to allow a range of nonhuman, CD4-expressing cells to Results support infection and cell fusion mediated by labora- tory-adapted HIV-1 envelope glycoproteins (Feng et al., A Subset of Chemokine Receptors Facilitates 1996). Feng et al. (1996) have suggested the name Infection by Macrophage-Tropic HIV-1 “fusin” for this protein, although a standardized nomen- To assess the efficiency with which HIV-1 viruses con- clature awaits identification of its natural function. Anti- taining different envelope glycoproteins mediate early bodies to HUMSTSR blocked cell fusion and infection events in HIV-1 infection, an env-complementation by laboratory-adapted HIV-1 isolates but not by macro- assay (Helseth et al., 1990; Thali et al., 1994) was utilized. phage-tropic primary viruses. While its natural ligand is Recombinant HIV-1 viruses were produced by cotrans- currently unknown, HUMSTSR exhibits sequence simi- fection of HeLa cells with two plasmids, pHXBH10⌬env- larity to the receptor for interleukin-8, an ␣ (CXC) chemo- CAT and pSVIIIenv. The pHXBH10⌬envCAT plasmid kine (Probst et al., 1992). It has also been observed that contains an HIV-1 provirus with a deletion in the env infection of macrophage-tropic primary HIV-1 isolates, gene and a replacement of the nef gene with a gene but not that of a laboratory-adapted isolate, could be encoding chloramphenicol acetyltransferase (CAT). Dif- inhibited by the ␤ chemokines RANTES, MIP-1␣, and ferent pSVIIIenv plasmids encoding the envelope glyco- MIP-1␤ (Cocchi et al., 1995). High endogenous expres- proteins derived from a laboratory-adapted HIV-1 iso- sion of these ␤ chemokines has been suggested to ac- late (HXBc2) and from macrophage-tropic primary HIV-1 count for the in vitro resistance to HIV-1 infection of isolates (Br20-4, ADA, and YU2) were used. The recom- CD4-positive T cells from uninfected individuals with binant viruses produced in the HeLa supernatants thus multiple sexual exposures to seropositive partners (Pax- contain different envelope glycoproteins, allowing an ton et al.,1996). This resistance was only seen for macro- assessment of the ability of these glycoproteins to medi- phage-tropic and not T cell line–tropic viruses and was ate a single round of infection. Control viruses lacking influenced by the structure of the third variable (V3) envelope glycoproteins were produced by transfecting gp120 region of the infecting virus. The available data HeLa cells with the pHXBH10⌬envCAT plasmid alone. suggest that at least one other host cell surface mole- An equal number of reverse transcriptase units of the cule besides CD4 and distinct from HUMSTSR facilitates recombinant viruses in the HeLa supernatants was incu- the entry of primary, macrophage-tropic HIV-1 isolates, bated with target cells. HeLa cells transfected with plas- and that this molecule might be influenced by interaction mids expressing human CD4 and various seven-trans- with ␤ chemokines. membrane-segment receptors were used as target ␤-Chemokine Receptors as Entry Cofactors for HIV-1 1137

cells. The efficiency of the early phase of virus infection was assessed by measurement of CAT activity in the HeLa target cells 60 hr following infection. Expression of human CD4 in HeLa cells was sufficient for efficient infection of these cells by a recombinant virus containing the laboratory-adapted HXBc2 enve- lopeglycoproteins (Figure 1; Table 1), as previously pub- lished (Brand et al., 1995). By contrast, infection of HeLa cells expressing CD4 by viruses with the macrophage- tropic ADA, YU2, and Br20-4 envelope glycoproteins was inefficient. Other studies have demonstrated that recombinant viruses with these envelope glycoproteins are able to infect human PBMC (Westervelt et al., 1991; Sullivan et al., 1995; Karlsson et al., 1996). These results are consistent with previous observations suggesting that macrophage-tropic primary HIV-1 isolates enter most immortalized cells inefficiently (Cheng-Mayer et al., 1988; Chesebro et al., 1991; Schuitemaker et al., 1991). Plasmids expressing the cDNAs of a number of che- mokine receptors and related molecules were cotrans- fected with the CD4-expressing plasmid into the HeLa cells. The level of CD4 expression on the surface of the HeLa cells was not affected by coexpression of the chemokine receptors examined (data not shown). As shown in Table 1, the expression of most of the seven- transmembrane receptors did not affect infection by the recombinant HIV-1 viruses. Expression of the CCR5 molecule resulted in significant enhancement of infec- tion by viruses with the ADA, YU2, and Br20-4 envelope glycoproteins, but had no effect on infection by the virus containing the HXBc2 envelope glycoproteins (Figure 1; Table 1). Expression of the CCR3 molecule also resulted in enhanced infection by the viruses with ADA and YU2 envelope glycoproteins. The magnitude of this effect (9- to 32-fold) was smaller than that seen for CCR5 (35- to 45-fold). CCR3 did not stimulate infection by the viruses with BR20-4 and HXBc2 envelope glycoproteins. The enhancing effects of CCR3 and CCR5 expression were not seen when human CD4 was not expressed in the HeLa target cells (Table 1). To examine whether the level of expression of the chemokine receptors on the surface of the transfected HeLa cells might have influenced the results, fluores- cence-activated cell sorter (FACS) analysis was used to verify the expression of a subset of the chemokine receptors (Table 1, legend). FACS analysis with a CCR3- directed monoclonal antibody revealed that cell surface expression of the wild-type CCR3 protein was inefficient (data not shown). This is consistent with previous at- tempts to express CCR3 in the context of a heterologous cell (Daugherty et al., 1996; Ponath et al., 1996b). To compare cell surface expression of CCR1, CCR3, and CCR5 directly, the CCR1F, CCR3F, and CCR5F proteins, which have identical amino-terminal epitope tags (Kunz Figure 1. CAT Activity in Transfected HeLa Cells Exposed to Re- et al., 1991), were studied. The presence of the epitope combinant HIV-1 Viruses tag on the CCR3F and CCR5F molecules only minimally HeLa cells expressing human CD4 and CD2 (A), CD4 and CCR1F affected the observed enhancement of infection by the (B), CD4 and CCR3 (C), or CD4 and CCR5 (D) were exposed to recombinant viruses containing either no envelope glycoproteins primary viruses (Table 1). Cell surface levels of CCR3F (None) or envelope glycoproteins of the ADA, Br20-4, YU2, or HXBc2 and CCR5F were approximately 83- and 24-fold lower, isolates. The results of the CAT assay performed on the HeLa cell respectively, than that of CCR1F in the transfected HeLa lysates are shown. cells, when background fluorescence was taken into account (Table 1, legend). Thus, the enhancement of Cell 1138

Table 1. CAT Activity in Transfected HeLa Cells Infected with Recombinant HIV-1

HIV-1 Envelope Glycoproteins

Molecules Expressed in Target Cells None ADA YU2 Br20-4 HXBc2 CD4 (ϩCD2 control) 0.29 1.5 1.1 0.79 53.5 CD4 ϩ CCR1 ND 1.3 1.1 0.79 ND CD4 ϩ CCR1F 0.25 2.0 2.0 1.1 51.9 CD4 ϩ CCR1F (sodium butyrate) ND ND 2.0 ND ND CD4 ϩ CCR2 0.30 2.1 1.7 0.85 54.2 CD4 ϩ CCR3 0.25 14.2 35.0 1.2 53.1 CCR3 ND 1.5 0.61 1.2 ND CD4 ϩ CCR3F 0.20 13.7 17.8 1.6 54.1 CD4 ϩ CCR3F (sodium butyrate) ND ND 32.7 ND ND CD4 ϩ CCR4F ND 1.1 1.5 0.67 ND CD4 ϩ CCR5 0.17 52.4 49.0 25.3 53.6 CCR5 ND 1.3 0.71 1.3 ND CD4 ϩ CCR5F 0.20 61.9 51.0 39.8 53.4 CD4 ϩ CCR5F (sodium butyrate) ND ND 81.9 ND ND CD4 ϩ IL8RA 0.28 1.5 0.84 0.64 57.0 CD4 ϩ IL8RB 0.41 1.9 1.3 0.64 55.1 CD4 ϩ HUMSTSR 0.19 1.1 0.68 0.45 53.0 CD4 ϩ Duffy antigen 0.08 0.41 0.33 0.22 53.4 CD4 ϩ EBI-1 ND 0.56 0.85 0.37 ND CD4 ϩ PAF-RF ND 0.92 0.37 0.79 ND CD4 ϩ FMLP-RF ND 2.0 1.0 2.3 ND CD4 ϩ C5aR ND 0.66 0.73 0.45 ND

HeLa target cells expressing the molecules shown were infected with recombinant HIV-1 viruses containing the envelope glycoproteins listed. In some of the experiments, the transfected HeLa target cells were treated with sodium butyrate prior to infection. CAT activity (percentage conversion of chloramphenicol to acetylated forms per unit of lysate) was determined. The values reported represent the mean value from duplicate experiments; standard deviation was less than 25% of the mean values. ND ϭ not determined. The surface expression of the CCR2, CCR3, IL8RA, IL8RB, and PAF-RF proteins was documented by FACS using specific monoclonal antibodies. The mean fluorescence intensities observed were as follows: background, 6.8; CCR2, 60.6; IL8RA, 425.9; IL8RB, 132.8; and PAF-RF, 26.3. The surface expression of the CCR1F, CCR3F, CCR4F, and CCR5F proteins was directly compared by using a monoclonal antibody reactive with the epitope tag (FLAG tag) on the amino terminus of each of these molecules. The mean fluorescence intensities observed were as follows: background, 9.9; CCR1F, 233.8; CCR3F, 12.6; CCR4F, 16.0; and CCR5F, 19.3. The mean fluorescence intensities observed for CCR1F and CCR3F following sodium butyrate treatment were 332.0 and 15.1, respectively. Background values were obtained by using only the secondary antibody (FITC-conjugated anti- mouse IgG) in the FACS analysis.

primary virus infection by CCR3 and CCR5 appears to ruses with primary, macrophage-tropic envelope glyco- be specific and is not merely a result of higher surface proteins. expression of these receptors. We wished to examine whether increasing the cell surface level of CCR3 and CCR5 might increase the Infection by Diverse HIV-1 Is Enhanced by CCR5 magnitude of the observed enhancement of infection. All of the envelope glycoproteins used in the experi- Prior to virus infection, transfected HeLa cells were incu- ments described above were derived from HIV-1 viruses bated with sodium butyrate, which has been shown to from phylogenetic clade B (Myers et al., 1994). To exam- enhance the transcriptional activity of the cytomegalovi- ine the generality of the observed enhancement, HeLa rus immediate early promoter used for chemokine re- cells transiently expressing CD4 and either CD2, CCR1F, ceptor expression on the pcDNA3 plasmid (Palermo et CCR3, or CCR5 were incubated with recombinant vi- al., 1991). FACS analysis indicated that sodium butyrate ruses containing envelope glycoproteins from a geo- treatment increased the surface level of CCR1F and graphically diverse set of primary HIV-1 isolates (Gao et CCR3F expression by approximately 1.5- to 1.9-fold, al., 1996; Karlsson et al., 1996). The results, shown in when background fluorescence was taken into account Table 2, indicate that CCR5 was able to enhance the (Table 1, legend). In cells expressing CCR3F and CCR5F, infection of a broader array of viruses than was CCR3. an increase in the level of infection by the virus with the The infection of all of the primary viruses was increased YU2 envelope glycoproteins resulted, whereas sodium in cells expressing CD4 and CCR5 relative to that seen butyratetreatment had no effect on infection bya control in cells expressing CD4 and CD2 or CD4 and CCR1F. recombinant virus containing the amphotropic murine Of the panel of viruses tested, only those containing leukemia virus (A-MuLV) envelope glycoproteins (Table the ADA and YU2 envelope glycoproteins infected HeLa 1 and data not shown). Sodium butyrate treatment of cells expressing CD4 and CCR3 more efficiently than HeLa cells transfected with plasmids expressing CD4 HeLa cells expressing CD4 and CD2. Recombinant vi- and CCR1F did not affect infection by the YU2 recombi- ruses containing laboratory-adapted (HXBc2) viral enve- nant virus. The results indicate that the cell surface levels lope glycoproteins did not infect HeLa cells expressing of CCR3 and CCR5 expression are limiting the magni- CD4 and either CCR3 or CCR5 more efficiently than they tude of the observed enhancement of infection by vi- infected control cells expressing CD4 and CD2 or CD4 ␤-Chemokine Receptors as Entry Cofactors for HIV-1 1139

Table 2. CAT Activity in Transfected HeLa Cells Infected with Diverse Recombinant HIV-1

Envelope Glycoproteins Molecules Expressed in Target Cells None ADA YU2 BR20-4 Br25-9 Rw20-5 Th966 TN243 HXBc2 A-MuLV CD4 (ϩCD2 control) 0.58 4.8 5.3 3.0 2.3 3.1 1.6 1.5 82.1 1742 CD4 ϩ CCR1F 0.33 5.7 5.0 3.6 3.5 8.4 1.4 2.5 131.7 1489 CD4 ϩ CCR3 0.35 45.9 70.5 3.0 5.0 5.4 1.3 1.5 111.3 1521 CD4 ϩ CCR5 0.36 102.3 103.9 49.8 11.6 70.4 16.7 16.6 104.9 812

HeLa cells expressing CD4 and chemokine receptors were incubated with recombinant viruses containing the designated envelope glycopro- teins, and CAT activity measured. The ADA and YU2 envelope glycoproteins were derived from macrophage-tropic primary HIV-1 viruses from North America (clade B). The Br20-4, Br25-9, Rw20-5, Th966, and TN243 envelope glycoproteins were derived from macrophage-tropic primary HIV-1 viruses. The phylogenetic classification and geographic origin of these viruses are as follows: Br20-4 (clade B, Brazil), Br25-9 (clade C, Brazil), Rw20-5 (clade A, Rwanda), Th966 (clade E, Thailand), and TN243 (clade E, Thailand) (Gao et al., 1996; Karlsson et al., 1996). The HXBc2 envelope glycoproteins were derived from a highly laboratory-adapted clade B HIV-1 isolate. The A-MuLV envelope glycoproteins were derived from the amphotropic murine leukemia virus (Landau et al., 1991). The values reported represent mean CAT activity per unit of cell lysate, as described in the Table 1 legend.

and CCR1F. Infection by control HIV-1 viruses pseu- expression on infection by the recombinant viruses used dotyped with the amphotropic murine leukemia virus in this study. Since most T cell line–tropic primary HIV-1 (A-MuLV) envelope glycoproteins (Landau et al., 1991) isolates and laboratory-adapted isolates enter CD4- was not increased by the expression of CCR3 and CCR5 positive HeLa cells efficiently (Chesebro et al., 1991), on the target cells. the use of the Cf2Th cells also allowed us to assess the effect of chemokine receptor expression on infection by Inhibition of CCR3-Dependent HIV-1 these types of viruses. The results in Figure 3 indicate Infection by Eotaxin that none of the recombinant HIV-1 viruses containing It has been reported that RANTES, MIP-1␣, and MIP- 1␤, the ligands for CCR5, inhibit the infection of primary HIV-1 isolates (Cocchi et al., 1995; Paxton et al., 1996). We wished to examine whether the binding of a ligand to CCR3 would affect the ability of this chemokine recep- tor to facilitate HIV-1 infection. HeLa-CD4 cells tran- siently expressing CCR3 were incubated with eotaxin, the major CCR3 ligand (Jose et al., 1994; Ponath et al., 1996a), prior to infection by recombinant viruses containing the YU2 or murine amphotropic (A-MuLV) envelope glycoproteins. Additional control HeLa-CD4 cells expressing CD2, CCR1F, or CCR5 were included in the assay. The data in Figure 2 indicate that eotaxin exhibited a dose-dependent inhibition of infection of HeLa-CD4 cells expressing CCR3 by YU2 recombinant viruses. No effect of eotaxin was observed, even at high concentrations, on infection by the recombinant virus with the A-MuLV envelope glycoproteins. No effect of eotaxin was observed on the infection of CCR5-express- ing HeLa-CD4 cells by the YU2 recombinant virus. These results indicate that, under circumstances where HIV-1 infection is dependent upon CCR3, eotaxin can inhibit the efficiency of this process.

Chemokine Receptors Facilitate CD4-Dependent HIV-1 Infection of Nonhuman Cells To examine whether CCR3 and CCR5 expression could facilitate HIV-1 infection of a nonhuman target cell, Cf2Th canine thymocytes were transfected with a plas- Figure 2. Effect of Eotaxin on CCR3-Mediated Enhancement of YU2 mid expressing human CD4 in combination with a plas- Recombinant Virus mid expressing either CD2, CCR3, or CCR5. A plasmid HeLa-CD4 cells transfected with plasmids expressing CD2, CCR1F, expressing HUMSTSR, which has been reported to facil- CCR3, or CCR5 were incubated for 1 hr at 37ЊC with increasing itate membrane fusion by laboratory-adapted HIV-1 iso- amounts of eotaxin. Recombinant HIV-1 viruses containing the enve- lates (Feng et al., 1996), was also included in this experi- lope glycoproteins of either the amphotropic murine leukemia virus (A-MuLV) or the YU2 macrophage-tropic primary isolate were added ment. Since HUMSTSR is expressed at high levels in to the cells. CAT activity in the cell lysates was assessed 72 hr later. HeLa cells (Feng et al., 1996), the use of different target The results of a single experiment, which was repeated with similar cells allowed an examination of the effect of HUMSTSR results, are shown. Cell 1140

the macrophage-tropic primary envelope glycoproteins (ADA, YU2), T cell line–tropic primary envelope glycopro- teins (89.6, ELI), or the laboratory-adapted (HXBc2) en- velope glycoproteins efficiently infected Cf2Th cells ex- pressing human CD4. A recombinant virus containing the A-MuLV envelope glycoproteins was able to infect the Cf2Th cells at a high level of efficiency. This was expected since all of the Cf2Th cells in the culture were potentially susceptible to infection by the virus with the A-MuLV envelope glycoproteins. By contrast, only the fraction of cells successfully transfected were poten- tially infectible by the viruses with HIV-1 envelope glyco- proteins. Expression of HUMSTSR in addition to CD4 facilitated infection by the HXBc2 and 89.6 recombinant viruses but did not affect infection by viruses with the ADA or YU2 envelope glycoproteins. A small positive effect of HUMSTSR expression was seen on infection by the ELI recombinant virus. These results are consis- tent with a published report indicating that HUMSTSR expression facilitated cell fusion directed by the enve- lope glycoproteins of laboratory-adapted HIV-1 but not of macrophage-tropic primary HIV-1 isolates (Feng et al., 1996). The results also demonstrate that HUMSTSR can be utilized by at least some T cell line–tropic primary envelope glycoproteins to facilitate infection. Coexpres- sion of CCR3 with human CD4 enhanced infection by the ADA and YU2 recombinant viruses, with smaller positive effects seen for the 89.6 and ELI recombinant viruses. CCR3 expression did not affect the efficiency of infec- tion by the virus with the HXBc2 envelope glycoproteins. Infection of the CD4-expressing Cf2Th cells by the ADA, YU2, and 89.6 recombinant viruses, but not by the ELI and HXBc2 recombinant viruses, was enhanced by the coexpression of CCR5. These results suggest that HUMSTSR can be utilized by some T cell line–tropic primary and laboratory-adapted HIV-1 isolates for infec- tion and that CCR3 and CCR5 can be utilized by some T cell line–tropic and macrophage-tropic primary isolates. The results are summarized in Table 3.

HIV-1 Envelope Glycoprotein Determinants of CCR3 and CCR5 Utilization A major, although not the sole, determinant of viral tro- pism is the primary structure of the third variable (V3) region of the HIV-1 gp120 glycoprotein (Cheng-Mayer et al., 1990; O’Brien et al., 1990; Hwang et al., 1991; Westervelt et al., 1991; Chesebro et al., 1992; Willey et al., 1994). To examine whether V3 structure influenced sensitivity of HIV-1 to the presence of CCR3 and CCR5 on the target cell surface, HeLa-CD4 cells expressing CCR3 or CCR5 were incubated with viruses containing chimeric gp120 envelope glycoproteins. These chimeric envelope glycoproteins are identical to that of the Figure 3. Effect of CCR3, CCR5, and HUMSTSR Expression on HXBc2 laboratory-adapted isolate, except that the V3 HIV-1 Infection of Cf2Th Canine Thymocytes loop is derived from the ADA and YU2 macrophage- Cf2Th canine thymocytes expressing human CD4 and CD2 (A), CD4 tropic primary isolates (Westervelt et al., 1992; Carrillo and CCR3 (B), CD4 and CCR5 (C), or CD4 and HUMSTSR (D) were et al., 1993). The ADA and YU2 V3 domains have been infected with recombinant viruses containing the indicated envelope glycoproteins. The CAT assay results are shown. shown to confer on chimeric envelope glycoproteins the ability to support HIV-1 infection of primary macro- phages (Westervelt et al., 1992). Table 4 shows that recombinant viruses containing the chimeric glycopro- teins with the ADA and YU2 V3 loops, in contrast to those ␤-Chemokine Receptors as Entry Cofactors for HIV-1 1141

Table 3. Properties of HIV-1 Envelope Glycoproteins Utilized in This Study

HIV-1 Envelope Syncytium Chemokine Glycoproteins Classification Induction Passage History Receptor Utilization Reference HXBc2 Laboratory- SI Extensive passage in HUMSTSR Fisher et al., Adapted T cell lines 1985 89.6 T cell line–tropic SI Homologous PBMC HUMSTSR, CCR5, Collman et al., primary CCR3 (low) 1992 ELI T cell line–tropic SI Homologous PBMC HUMSTSR (low), Alizon et al., primary CCR3 (low) 1986; Peden et al., 1991 ADA Macrophage–tropic NSI Heterologous CCR3, CCR5 Gendelman et primary primary al., 1988 macrophages YU2 Macrophage–tropic NSI Unpassaged CCR3, CCR5 Li et al., 1991 primary

The ability of the molecularly cloned envelope glycoproteins used in this study to induce the formation of syncytia in immortalized T cell lines is indicated. SI ϭ syncytium-inducing, NSI ϭ non-syncytium-inducing. The passage history of the virus prior to molecular cloning of the env gene is indicated. The chemokine-receptor utilization is derived from the data in Figure 3.

containing the parental HXBc2 envelope glycoproteins, HeLa cells expressing the HXBc2 envelope glycopro- were able to infect HeLa-CD4 cells more efficiently when teins formed syncytia with HeLa-CD4 cells (data not either CCR3 or CCR5 was expressed on the target cell. shown), consistent with the expression of endogenous Substitution of the YU2 V1/V2 variable loops into the HUMSTSR in this cell line (Feng et al., 1996). The ADA HXBc2 envelope glycoproteins did not increase the effi- and YU2 envelope glycoproteins, by contrast, did not ciency of infection of HeLa-CD4 target cells expressing efficiently mediate the formation of syncytia with the CCR3 or CCR5, compared with the cells expressing CD4-positive HeLa cells expressing the CCR1 protein. the CD2 control protein. These results indicate that the Syncytium formation directed by the chimeric HXBc2 structure of the gp120 V3 loop can influence the ability (ADA-V3) and HXBc2 (YU2-V3) envelope glycoproteins of HIV-1 viruses to respond to the presence of these was also inefficient with CCR1-expressing HeLa-CD4 chemokine receptors in the target cells. target cells. Expression of CCR3 in addition to CD4 on the HeLa cells resulted in syncytium formation directed by the YU2 envelope glycoproteins. The expression of Cell–Cell Fusion Is Influenced by CCR3 and CCR5 CCR5 on the HeLa-CD4 cells allowed the formation of Our results indicate that an envelope glycoprotein-spe- syncytia with cells expressing the ADA, YU2, and chime- cific process early in the HIV-1 life cycle is influenced ric envelope glycoproteins. The number of syncytia by the expression of CCR3 and CCR5 on the target cell. formed by the HXBc2 envelope glycoproteins was not To examine whether the membrane fusion process is affected by CCR3 or CCR5 expression on the CD4-posi- enhanced by the presence of these chemokine recep- tive HeLa cells (data not shown). Syncytium formation tors, we utilized an assay measuring HIV-1 envelope in this assay was dependent upon gp120 binding to glycoprotein-mediated syncytium formation. In this CD4, since the OKT4a anti-CD4 monoclonal antibody, assay, HeLa cells expressing different HIV-1 envelope which blocks gp120-CD4 interaction (McDougal et al., glycoproteins were cocultivated with either mock- 1986), inhibited the formation of syncytia. These results transfected HeLa-CD4 cells or HeLa-CD4 cells express- indicate that expression of CCR3 and CCR5 on CD4- ing CCR1, CCR3, or CCR5. Figure 4 shows that the positive target cells can enhance fusion events medi- number of syncytia observed when no envelope glyco- ated by macrophage-tropic primary virus envelope gly- proteins were expressed in the HeLa cells was minimal. coproteins. The results also indicate that the HIV-1

Table 4. CAT Activity in Transfected HeLa-CD4 Cells Infected with Recombinant Viruses Containing Chimeric HIV-1 Envelope Glycoproteins

Envelope Glycoproteins

Molecules Expressed in HXBc2 HXBc2 HXBc2 HeLa-CD4 Target Cells ADA YU2 HXBc2 (ADA-V3) (YU2-V3) (YU2-V1/V2)

CD2 1.4 0.88 546 1.3 0.87 914 CCR3 19.0 73.4 542 40.8 5.9 1016 CCR5 ND ND 543 37.6 40.9 417

HeLa-CD4 cells expressing chemokine receptors were incubated with recombinant viruses containing wild-type or chimeric envelope glycopro- teins, and CAT activity measured. The HXBc2 (ADA-V3) and HXBc2 (YU2-V3) envelope glycoproteins are identical to the HXBc2 envelope glycoprotein except for a substitution of the ADA or YU2 V3 loop in the gp120 glycoprotein (Westervelt et al., 1992; Carillo et al., 1993). The HXBc2 (YU2-V1/V2) chimeric envelope glycoprotein contains a substitution of the V1/V2 loops from the YU2 virus into the HXBc2 gp120 glycoprotein. ND ϭ not determined. The values shown in the table are from a representative experiment, which was repeated with comparable results. Cell 1142

Figure 4. Effect of Chemokine Receptor Expression on HIV-1 Envelope Glycoprotein-Directed Syncytium Formation HeLa cells expressing either no envelope glycoprotein (None) or the ADA, YU2, HXBc2 (ADA-V3), and HXBc2 (YU2-V3) envelope glycoproteins were cocultivated with HeLa-CD4 expressing CCR1, CCR3, or CCR5. In one set of experiments, 2 ␮g/ml of the OKT4a antibody (Ortho Pharmaceuticals, Inc.) was added at the beginning of the cocultivation. After 12 hr, the syncytia in the wells were counted. The results of a single experiment are shown. The experiment was repeated with comparable results. gp120 V3 loop sequence determines the ability of the (Daugherty et al., 1996; Kitaura et al., 1996; Ponath et envelope glycoproteins to utilize CCR5 as a fusion co- al., 1996b). The latter observation suggests that CCR3 factor. could not be the sole factor facilitating the infection of primary HIV-1 isolates, all of which replicate in PBMC. Discussion While the involvement of CCR5 is likely to be relevant to a greater variety of HIV-1 target cells, CCR3 may The results presented herein indicate that, in addition play an important role in a limited number of cell types. to CD4, members of the chemokine receptor family play Eosinophils, which express high levels of CCR3 (Daugh- critical roles in early events in HIV-1 infection. The par- erty et al., 1996; Kitaura et al., 1996; Ponath et al., 1996b), ticular chemokine receptors utilized by HIV-1 variants also express CD4 and have been reported to be in- differ, depending upon previously characterized differ- fectible by HIV-1 (Freedman et al., 1991; Weller et al., ences in target cell preference. The ability of laboratory- 1995). In one of these studies (Freedman et al., 1991), adapted HIV-1 viruses to replicate in immortalized CD4- two primary HIV-1 isolates, but not a laboratory-adapted positive cell lines has been shown to involve an orphan virus, were able to replicate in bone marrow–derived receptor referred to as fusin, HUMSTSR, LESTR, or eosinophils. It is not known whether the ability of mono- LCR1 (Feng et al., 1996). Our results confirm the involve- cytes to respond to high concentrations of eotaxin (Po- ment of HUMSTSR in infection by laboratory-adapted nath et al., 1996a) indicates a low level of expression of HIV-1 and demonstrate a role for this molecule in infec- CCR3 on these cells, or the presence of another low tion by some primary, T cell line–tropic HIV-1 isolates. affinity receptor. MCP-3, one of the CCR3 ligands, is an Our results indicate that the clinically relevant, macro- important chemotactic factor for dendritic cells (Sozzani phage-tropic HIV-1 can use other members of the chem- et al., 1995), which have been suggested to play an okine receptor family, such as CCR3 and CCR5, to facili- important role in HIV-1 transmission across mucosal tate infection. barriers (Spira et al., 1996). The expression of CCR3 on Although the tissue and cell-type distribution of CCR3 these and other potential HIV-1 target cells merits further and CCR5 is not completely characterized, current data investigation. are consistent with the hypothesis that these molecules The expression of CCR3 significantly enhanced infec- contribute to in vivo infection by macrophage-tropic pri- tion by a smaller subset of primary HIV-1 than did CCR5 mary HIV-1 variants. The tissue distribution of CCR5 has expression. Although infection mediated by several of beenreported to be restricted to KG-1A promyeloblastic the primary HIV-1 envelope glycoproteins was not de- cells (Samson et al., 1996), but more recent data suggest tectably affected by CCR3 expression, significant CCR3 that it is expressed in both CD4-positive and CD8-posi- effects were observed for the YU2 and ADA envelope tive human PBMC, as well as in cells of the myeloid glycoproteins. This result suggests that heterogeneity lineage (C. G., unpublished data and Raport et al., 1996). in chemokine receptor utilization may occur even among The latter distribution is consistent with that expected macrophage-tropic primary HIV-1 isolates. It is perhaps based upon the known host cell range of primary HIV-1 relevant that, while all primary HIV-1 isolates are more isolates. The expression of CCR3 appears to be more resistant to antibody neutralization than are laboratory- restricted, with high levels of expression in eosinophils adapted viruses (Montefiori et al., 1991; Bou-Habib et and little expression in peripheral blood T lymphocytes al., 1994; Burton et al., 1994; Mascola et al., 1994; Moore ␤-Chemokine Receptors as Entry Cofactors for HIV-1 1143

et al., 1995; Sullivan et al., 1995; Wrin et al., 1995; Karls- simplest model for post-CD4 binding events in HIV-1 son et al., 1996), infection by the ADA and YU2 viruses entry would involve a direct interaction between the viral is actually enhanced by neutralizing antibodies (Sullivan envelope glycoproteins and the chemokine receptors. et al., 1995). It is unknown whether antibody-mediated The variability of gp120 tropism determinants, in particu- enhancement is related to utilization of CCR3, but the lar the V3 loop, among HIV-1 strains contrasts with the magnitude of both antibody and CCR3 enhancement minimal polymorphism observed in particular chemo- was greatest on viruses containing the YU2 envelope kine receptors. Our data indicate that infection by geo- glycoproteins. These observations raise the possibility graphically and phylogenetically diverse HIV-1 isolates that a subset of primary HIV-1 exhibits previously unsus- can be facilitated by the same CCR5 molecule. If direct pected properties allowing continued replication in par- envelope glycoprotein-chemokine receptor interaction ticular cell types in the presence of neutralizing antibod- occurs, it may involve conserved structures on the ies. The YU2 sequences were directly cloned into phage gp120 variable loops not apparent from inspection of vectors from the central nervous system of an infected primary amino acid sequences. This situation may be patient, thus avoiding even minimal passage on PBMC analogous to the binding of apparently diverse chemo- (Li et al., 1991). Isolation of HIV-1 on peripheral blood T kines by the same chemokine receptor. Alternatively, lymphocytes, which do not express CCR3, in the ab- conserved envelope glycoprotein structures influenced sence of neutralizing antibodies may remove selection indirectly by variable loop configurations may directly pressure for viruses that can utilize these molecules to interact with the chemokine receptors. While CCR3 and enhance infection. Establishment of different in vitro CCR5 are closely related among the chemokine recep- culture systems may allow the identification of addi- tors (Daugherty et al., 1996; Ponath et al., 1996b; Raport tional HIV-1 isolates that can efficiently utilize CCR3. et al., 1996; Samson et al., 1996), the relationship of The contribution of CCR3 to primary HIV-1 infection of either of these molecules to CCR1, which did not affect different target cells in vivo and the relationship between HIV-1 infection in our hands, is even greater. Again, CCR3 use and resistance to neutralizing antibodies will simple inspection of primary sequences does not reveal be evaluated in future studies. determinants unique to CCR3 and CCR5 that might be The involvement of receptors for the ␤ chemokines in targets for HIV-1 interaction. An alternative model is that HIV-1 infection explains the sensitivity of macrophage- the chemokine receptors affect the target membrane tropic primary HIV-1 isolates, but not laboratory- and/or CD4 in ways conducive to entry by viruses with adapted isolates, to inhibition by RANTES, MIP-1␣, and particular envelope glycoprotein configurations, without MIP-1␤ (Cocchi et al., 1995). Both CCR3 and CCR5 have directly contacting viral components. It is also possible been shown to be responsive to RANTES (Daugherty et that G protein–mediated signaling plays a role in HIV-1 al., 1996; Ponath et al., 1996b; Samson et al., 1996). The infection. Additional studies should distinguish among increased HIV-1 inhibitory activity of RANTES compared these possibilities. with MIP-1␣ and MIP-1␤ suggests that CCR5 may not The involvement of G protein–coupled receptors in be solely responsible for mediating this inhibition, since two other instances of infection with pathogens has CCR5 has been reported to exhibit greater sensitivity been reported. The Duffy antigen receptor, which binds to MIP-1␣ than to RANTES (Samson et al., 1996). The both ␣ and ␤ chemokines, facilitates invasion by the involvement of another chemokine receptor, such as malarial parasite, Plasmodium vivax (Chaudhuri et al., CCR3, which is responsive to RANTES but not to MIP- 1993; Horuk et al., 1993). Similarly, the progression from 1␣ or MIP-1␤, in the HIV-1 inhibitory effect could explain colonization to infection with Streptococcus pneumon- the data. Our data on eotaxin inhibition of CCR3-medi- iae is facilitated by expression of the platelet-activating ated HIV-1 infection suggest that suppression of virus factor receptor (Cundell et al., 1995). infection may be a general consequence of ligand bind- The ␤-chemokine receptors identified here may repre- ing to chemokine receptors that are specifically used sent important host components that specify suscepti- by particular HIV-1 strains. The mechanism by which bility to HIV-1 infection or, in already infected individuals, chemokines exert their inhibitory effects on HIV-1 entry determine viral burden and rate of disease progression. maybe complex, involving receptorblockade, desensiti- Endogenous levels of RANTES, MIP-1␣, and MIP-1␤ zation, sequestration or internalization, phosphoryla- expression in CD4-positive lymphocytes were higher tion, or change in affinity state through G-protein uncou- in some individuals that remained uninfected despite pling (von Zastrow and Kobilka, 1992; Barak et al., 1994). multiple sexual exposures to HIV-1 infected partners Further work will be required to distinguish among these (Paxton et al., 1996). Additional contributions to patho- possibilities. genic processes may occur as a result of inappropriate The ability of CCR3 and CCR5 to enhance both syncy- signaling events triggered by direct interaction between tium formation and the early phase of HIV-1 infection viral components and the ␤-chemokine receptors. A bet- suggests that these molecules facilitate virus binding to ter understanding of the interaction of HIV-1, ␤ chemo- the target cell and/or membrane fusion. The structure kines, and their receptors may clarify the contribution of the gp120 V3 loop, previously shown to specify target of these elements to virus transmission and pathogenic cell–dependent membrane fusion efficiency (Cheng- outcome and may suggest approaches for intervention. Mayer et al., 1990; O’Brien et al., 1990; Hwang et al., 1991; Ivanoff et al., 1991; Westervelt et al., 1991, 1992; Experimental Procedures Bergeron et al., 1992; Chesebro et al., 1992), determined Plasmids the ability of the viral envelope glycoproteins to utilize The pHXBH10⌬envCAT and pSVIIIenv plasmids used to produce CCR3 and CCR5 as accessory factors for entry. The recombinant HIV-1 virions have been previously described (Helseth Cell 1144

et al., 1990; Thali et al., 1994). The pCD4 plasmid expressing full- For some of the experiments, the level of chemokine receptor length human CD4 has been described (Brand et al., 1995). The SV- expression on the transfected HeLa cells was measured by FACS A-MLV-Env plasmid expressing the amphotropic murine leukemia analysis 60 hr following transfection. The antibody (anti-FLAG M2, virus envelope glycoproteins was obtained from Dr. Dan Littman Kodak) against the epitope tag (FLAG tag) was used for analysis (Landau et al., 1991). The derivation and construction of the pSVIII- of CCR1F, CCR3F, CCR4F, and CCR5F expression. Monoclonal env plasmids expressing the envelope glycoproteins from various antibodies were used to detect surface expression of CCR2, PAF- strains of HIV-1 have been described (Sullivan et al., 1995; Gao et RF, IL8RA, and IL8RB. al., 1996; Karlsson et al., 1996). The chimeric HXBc2 (YU2-V3) and In some of the experiments, Cf2Th canine thymocytes were used HXBc2 (ADA-V3) env constructs were kindly supplied by Lee Ratner, as target cells. The Cf2Th cells were transfected by the calcium and were designated HY (V3A ϩ V3B) and HA (V3A ϩ V3B) in a phosphate technique with 10 ␮g of the pCD4 plasmid and 25 ␮gof previouspublication (Carrillo et al., 1993). The chimeric HXBc2 (YU2- the pcDNA3 plasmid expressing chemokine receptors or, as a con- V1/V2) env genes were created by substituting the DraIII to StuI trol, with 10 ␮g of the pCD4 plasmid and 25 ␮g of the pCDM8 fragment of the YU2 env gene into the corresponding segment (nu- plasmid expressing CD2 (see above). Approximately 72 hr after cleotides 6619–6901) of the HXBc2 env gene. transfection, the Cf2Th cells were incubated with recombinant HIV-1 The cDNAs encoding the chemokine receptors were cloned into and used for measurement of CAT activity as described above. the pcDNA3 vector (Invitrogen) for expression. Full-length cDNAs for CCR1 (Neote et al., 1993), CCR2A and CCR2B (Charo et al., Eotaxin Inhibition of HIV-1 Infectivity 1994), CCR3 (Daugherty et al., 1996; Ponath et al., 1996b), CCR4 Eotaxin was chemically synthesized by Dr. Ian Clark-Lewis and puri- (Power et al., 1995), and CCR5 (Samson et al., 1996) were cloned fied by high-pressure liquid chromatography after renaturation (Po- by polymerase chain reaction, sequenced and shown to encode nath et al., 1996a). Recombinant HIV-1 containing the YU2 and receptors capable of binding ligands following transfection. CCR1 A-MuLV envelope glycoproteins were produced in HeLa cells as was shown to bind MIP-1␣, RANTES, and MCP-3; CCR2 bound described above. HeLa-CD4 (clone 1022) cells, transfected either MCP-1 and MCP-3; CCR3 bound eotaxin, RANTES, and MCP-3; with the pCDM8 plasmid expressing CD2 or with the pcDNA3 plas- and CCR5 bound MIP-1␣, MIP-1␤, and RANTES. Plasmids encoding mid expressing chemokine receptors, were used as target cells. IL8RA, IL8RB, PAF-RF, FMLP-RF, and C5aR were previously de- The target cells, in 1 ml medium, were incubated with different scribed by the Gerard laboratory (Kunz et al., 1991; Gerard and concentrations (0–60 nM) of eotaxin for 90 min at 37ЊC. Medium Gerard, 1994). The HUMSTSR and Duffy antigen cDNAs were gener- was then removed and the cells were resuspended in 1 ml medium ously provided by Dr. Kuldeep Neote. The EBI-1 expressor plasmid containing recombinant virus (15,000 reverse transcriptase units). (Birkenbach et al., 1993) was obtained from Dr. Elliott Kieff. Eotaxin was added to the virus–cell mixture at the original concen- The CCR1, CCR3, CCR4, and CCR5 proteins were also expressed tration. After 12 hr at 37ЊC, the cells were washed and returned to as fusion proteins containing an epitope tag (MDYKDDDDK) (FLAG the incubator. After an additional 48 hr at 37ЊC, the cells were lysed tag, IBI-Kodak) at the amino terminus. These fusion proteins are and used for measurement of CAT activity. referred to as CCR1F, CCR3F, CCR4F, andCCR5F, respectively.The platelet activating factor receptor and the f-Met-Leu-Phe receptor contain the identical amino-terminal epitope tag, and are referred Syncytium Formation Assay to as PAF-RF and FMLP-RF, respectively. Envelope glycoprotein-expressing HeLa cells were derived by co- transfection of HeLa cells with pSVIIIenv plasmids expressing HIV-1 Cell Lines envelope glycoproteins and a plasmid encoding the HIV-1 Tat pro- HeLa cells were grown in Dulbecco’s modified Eagle’s medium con- tein (Helseth et al., 1990). Target cells were derived by transfection taining 10% fetal bovine serum and antibiotics. HeLa-CD4 (clone of HeLa-CD4 (clone 1022) cells with plasmids expressing either 1022) cells were obtained from Dr. Bruce Chesebro through the CCR1, CCR3, or CCR5. The envelope glycoprotein-expressing and National Institutes of Health AIDS Research and Reference Reagent target HeLa cells were detached, 48 hr after transfection, from the Program. The Cf2Th canine thymocyte line was obtained from the tissue culture plates using 5 mM EDTA. Cells were replated at a American Type Culture Collection (ATCC CRL 1430) and was propa- ratio of ten target cells to one envelope glycoprotein-expressing gated in Dulbecco’s modified Eagle’s medium containing 10% fetal cell and incubated at 37ЊCin5%CO2. The number of syncytia in bovine serum. the wells was counted 12 hr later. Control experiments were per- formed in which 2 ␮g/ml OKT4a (Ortho Pharmaceuticals, Inc.) was included at the time of replating. An additional control using the Env-Complementation Assay pCEP4 plasmid (Invitrogen), which does not express any envelope HeLa cells were cotransfected by the calcium phosphate method glycoproteins, was performed to assess background levels of syn- (Cullen, 1989) either with 15 ␮g pHXBH10⌬envCAT alone or with cytia. 15␮g pHXBH10⌬envCAT and 3 ␮g pSVIIIenv or SV-A-MLV-Env to produce recombinant virions, as previously described (Thali et al., 1994; Karlsson et al., 1996). HeLa cells to be used as target cells Acknowledgments were plated at 7 ϫ 105 cells per 100 mm dish, cultured overnight, and then transfected by the calcium phosphate method with 10 ␮g We thank Drs. Heinrich Go¨ ttlinger, Lee Ratner, Ellis Reinherz, Be- pCD4 and 25 ␮g pcDNA3 expressing chemokine receptors. Control atrice Hahn, Elliott Kieff, Gunilla Karlsson, and Dan Littman for gifts HeLa cells were transfected with 10 ␮g pCD4 and 25 ␮g pCDM8 of plasmids. We thank Dr. Bruce Chesebro for the HeLa-CD4 (clone expressing the CD2 protein, which has been shown to have no effect 1022) line, and acknowledge the National Institutes of Health AIDS on HIV-1 infection (H. Choe and J. Sodroski, unpublished data). The Research and Reference Reagent Program for providing these cells. pCDM8 plasmid expressing CD2 was a gift from Dr. Ellis Reinherz. We thank Ian Clark-Lewis for the synthesis of eotaxin. We thank Dr. The HeLa target cells were detached, 60 hr after transfection, from Wolfgang Hofmann for helpful advice. We thank Ms. Lorraine Rabb the tissue culture dish by treatment with phosphate-buffered saline and Ms. Jan Welch for manuscript preparation and Ms. Amy Emmert and 5 mM EDTA. The cell suspension was diluted in medium, with for artwork. This work was supported by a grant to J. S. from the one aliquot used for FACS analysis and the remaining aliquots re- National Institutes of Health (AI24755) and by a Center for AIDS plated into 6-well plates for infection. The level of CD4 expressed Research grant to the Dana-Farber Cancer Institute (AI28691). Dana- on the cell surface was measured by flow cytometry, using the FITC- Farber Cancer Institute is also the recipient of a Cancer Center grant conjugated OKT4 antibody reactive with CD4 domain 3 (McDougal from the National Institutes of Health (CA 06516). N. P. G. and C. et al., 1986). Approximately 8 hr after replating, cells were infected G. were supported by NIH grants HL51366 and AI36162, as well as by incubation with recombinant virions (20,000 cpm of reverse tran- by the Rubenstein/Cable Fund at the Perlmutter Laboratory. B. R. scriptase activity) in 1 ml of medium. After overnight incubation at is a Scholar of the Leukemia Society of America and is supported by NIH grant CA53091. This work was made possible by gifts from 60ف 37ЊC, additional medium was added to the cells. After a total of hr of incubation of the virus–cell mixture at 37ЊC, the cells were the late William McCarty-Cooper, from the G. Harold and Leila Y. lysed and used for determination of CAT activity. Mathers Charitable Foundation, and from the Friends 10. ␤-Chemokine Receptors as Entry Cofactors for HIV-1 1145

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