Identification of a Chemokine Receptor Encoded by Human Cytomegalovirus as a Cofactor for HIV-1 Entry

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Science  20 Jun 1997:
Vol. 276, Issue 5320, pp. 1874-1878
DOI: 10.1126/science.276.5320.1874


The human cytomegalovirus encodes a β-chemokine receptor (US28) that is distantly related to the human chemokine receptors CCR5 and CXCR4, which also serve as cofactors for the entry into cells of human immunodeficiency virus–type 1 (HIV-1). Like CCR5, US28 allowed infection of CD4-positive human cell lines by primary isolates of HIV-1 and HIV-2, as well as fusion of these cell lines with cells expressing the viral envelope proteins. In addition, US28 mediated infection by cell line–adapted HIV-1 for which CXCR4 was an entry cofactor.

Human immunodeficiency virus infects cells by a process of membrane fusion that is mediated by its envelope glycoproteins (gp120-gp41, or Env) and is generally triggered by the interaction of gp120 with two cellular components: CD4 and a coreceptor belonging to the chemokine receptor family (1). The coreceptor for HIV-1 strains adapted to replication in CD4+cell lines (TCLA strains) was identified by a genetic complementation approach and named fusin (2); however, it was later shown to be an α- (or CXC) chemokine receptor and designated CXCR4 (3). The isolation of fusin and the antiviral activity of certain β- (or CC) chemokines (4) led to the demonstration that the β-chemokine receptor CCR5 is the principal coreceptor for primary HIV-1 strains (5-8). In addition to CCR5, certain primary HIV-1 strains (dual tropic) use CXCR4 (9), or CXCR4 and CCR2b (8), as a coreceptor, whereas others (macrophage tropic) can use CCR3 (7, 10). The essential role of CCR5 is nevertheless indicated by the resistance to HIV-1 infection of individuals with defective CCR5 alleles (11). The CCR5 and CXCR4 coreceptors are also used by HIV-2 and the related simian immunodeficiency viruses (12, 13).

Several homologs of chemokine receptors are encoded by herpesviruses (14); in particular, by the US27, US28, and UL33 open reading frames (ORFs) of the human cytomegalovirus (CMV) (15). In fibroblasts infected experimentally, these ORFs were transcribed at a high rate after viral DNA replication (16), but their pattern of expression in vivo and their role in the life cycle of CMV are unknown. The product of the US28 ORF (here referred to as US28) is a functional receptor for several β-chemokines, including the CCR5 ligands RANTES (regulated on activation, normal T expressed and secreted), macrophage inflammatory protein–1α (MIP-1α), and MIP-1β (17). We therefore investigated whether US28 exhibits HIV coreceptor activity.

The human glioma-derived cell line U373MG-CD4 is naturally resistant to HIV-1 entry and to fusion with Env+cells and is stably transfected with a long terminal repeat (LTR)–lacZ construct inducible by the HIV-1 transactivator Tat (18). Cells infected by HIV, or syncytia formed with Tat+Env+ cells, can therefore be detected with high specificity by an in situ β-galactosidase assay (blue staining with the X-Gal substrate), as described previously (12, 18). U373MG-CD4 cells were transfected with expression vectors encoding US28, CCR5, or CXCR4 (19) and then tested for their ability to form syncytia with various Env+ cells (Fig.1A). As expected, U373MG-CD4 cells expressing CXCR4 formed syncytia with HeLa–Env/LAI cells (20) stably expressing Env from HIV-1LAI (TCLA), whereas U373MG-CD4 cells expressing CCR5 formed syncytia with HeLa–Env/ADA cells stably expressing Env from the primary macrophage-tropic HIV-1ADA(21). Both CXCR4 and CCR5 allowed the formation of syncytia with cells chronically infected with HIV-2ROD(22). The expression of US28 allowed fusion of U373MG-CD4 cells with each of the three types of Env+ cells. Similar results were obtained in infection assays (Fig. 1B). The macrophage-tropic HIV-1ADA and HIV-1Jr-CSF (23) infected U373MG-CD4 cells expressing CCR5 or US28, but not those expressing CXCR4. The TCLA strains HIV-1LAI and HIV-1NDK(24) infected cells expressing CXCR4 or US28, but not those expressing CCR5. The U373MG-CD4 cells were not totally resistant to infection with HIV-2ROD, but the efficiency of infection was markedly increased by expression of either US28, CXCR4, or CCR5. In these experiments, US28 behaved as a coreceptor for primary HIV-1 strains, and for HIV-2ROD, with an efficiency similar to that of CCR5. In addition, US28 mediated infection by TCLA HIV-1 strains, although less efficiently than CXCR4.

Figure 1

HIV coreceptor activity of CXCR4, CCR5, and US28 expressed in the human cell line U373MG-CD4. U373MG-CD4 cells (LTR-lacZ +) were harvested 24 hours after transfection and transferred to 24-well plates for coculture (1:1 ratio) with HeLa–Env/ADA, HeLa–Env/LAI, or HIV-2ROD–infected cells (A), or for infection with the HIV-1 strains ADA, Jr-CSF, LAI, or NDK (same inoculums as in Table 1), or with HIV-2ROD (50 ng of p24 per well) (B). Cells were fixed and stained with X-Gal 20 hours after initiation of coculture or 40 hours after infection. Data represent numbers of blue-stained foci per well (mean of two experiments, with independent transfections). Error bars represent the range between mean and maximal values.

The coreceptor activity of US28 was tested in another CD4+human cell line, HeLa-P4, also stably transfected with an LTR-lacZ construct (25). These cells are permissive to infection by TCLA and dual-tropic HIV-1 and HIV-2, but not to infection by primary strains with a macrophage-tropic or non–syncytium-inducing (NSI) phenotype. Accordingly, they formed syncytia, detectable by staining with X-Gal, on coculture with HeLa–Env/LAI but not with HeLa–Env/ADA cells (Fig.2A). Fusion with HeLa–Env/ADA was readily observed when HeLa-P4 cells were transfected with the US28 expression vector (Fig.2A), although a higher number of syncytia was generally apparent on expression of CCR5 (Fig. 2B). In contrast, fusion with HeLa–Env/ADA cells was not observed when HeLa-P4 cells were transfected with expression vectors encoding CXCR4 (Fig. 2B) or other chemokine receptors—in particular, CCR1 and the Duffy antigen—or with expression vectors containing the US27 or UL33 ORFs (26). Also, fusion with Env+ cells was not detected when the US28 or CCR5 vectors were transfected into CD4-negative LTR-lacZHeLa cells (25).

Figure 2

HIV-1 coreceptor activity of CCR5 and US28 in HeLa-P4 cells (CD4+LTR-lacZ +). (A) HeLa-P4 cells that had been mock-transfected (a and b) or transfected with the US28 vector (c and d) were cocultured with HeLa–Env/LAI cells (a and c) or HeLa–Env/ADA cells (b and d), and then stained with X-Gal, as described in Fig. 1A. (B) Syncytia formation in cocultures of HeLa–Env/ADA cells and HeLa-P4 cells transfected with expression vectors encoding CCR5, US28, CXCR4, or their epitope-tagged forms (T-US28, T-CCR5, and T-CXCR4). The experiment was performed as described in Fig. 1A. Data represent numbers of blue-stained foci per well.

To facilitate detection of US28, CCR5, and CXCR4 at the cell surface, we engineered their NH2-termini to express a c-MYC epitope (27). Transfection of HeLa-P4 cells with vectors encoding the epitope-tagged forms of US28 or CCR5 allowed fusion with HeLa–Env/ADA cells (Fig. 2B). The epitope tag markedly reduced the activity of US28, but had a lesser effect on the activity of CCR5 (Fig. 2B). Flow cytometry of transfected HeLa-P4 cells (28) revealed that the surface expression of epitope-tagged US28 was reduced relative to that of the tagged forms of CCR5 or CXCR4 (Fig. 3). Differences in the surface expression of epitope-tagged chemokine receptors have been observed previously (7). Thus, modification of the NH2-terminal domain of US28 may reduce HIV coreceptor activity or have an indirect effect on its transport to or stability at the cell surface.

Figure 3

Detection of epitope-tagged chemokine receptors by flow cytometry. HeLa-P4 cells were cotransfected with the EGFP-N1 vector encoding the green fluorescent protein (GFP) and either an expression vector encoding epitope-tagged CXCR4, CCR5, or US28 or the pCDNA-tag vector (control). Cells were detached 36 hours later and stained with the 9E10 tag-specific monoclonal antibody and phycoerythrin (PE)-conjugated secondary antibodies. (A) Analysis for red (xaxis) and green (y axis) fluorescence. The percentage of PE-positive cells (PE fluorescence, >30 arbitrary units) is indicated. (B) Similar analysis for 104cells selected for green fluorescence (>100 arbitrary units). The percentage of PE-stained cells (>50 arbitrary units) and mean PE fluorescence intensity are also indicated.

HeLa-P4 cells were stably transfected with the CCR5 or US28 vectors (29), and clones were selected for their ability to form syncytia with HeLa–Env/ADA cells (Fig. 4A). Unlike the parental cells, HeLa-P5 (expressing CCR5) and HeLa-P6 (expressing US28) cells could be infected by HIV-1ADA, HIV-1Jr-CSF, and two primary NSI strains (VEN and BX01), as detected by staining cells with X-Gal (Table 1) or, in the case of HIV-1ADA, by polymerase chain reaction (PCR) amplification of the viral DNA. The infection of HeLa-P5 cells was more efficient for all strains tested. However, Northern (RNA) blot analysis suggested that CCR5 is expressed at a higher level in this cell line relative to US28 in HeLa-P6 cells (Fig. 4B). The infection of HeLa-P5 and HeLa-P6 cells with HIV-1LAI was less efficient compared with infection of HeLa-P4 cells (Table 1); this difference was apparently not due to a lower abundance of CXCR4 mRNA in the former cell types (Fig. 4B).

Figure 4

Stable expression of CCR5 and US28 in HeLa-P4 cells. (A) Formation of syncytia in cocultures of HeLa–Env/ADA cells with either HeLa-P4 cells or cell lines derived by stable transfection of HeLa-P4 cells with CCR5 (HeLa-P5) or US28 (HeLa-P6) expression vectors. Cocultures (1:1) were stained with X-Gal after 20 hours. Data represent numbers of blue-stained syncytia (mean of two wells). (B) Detection of CXCR4, CCR5, US28, and β-actin (control) mRNA by Northern blot hybridization of total RNA (10 μg per lane) from HeLa-P4, HeLa-P5, and HeLa-P6 cells, as indicated. The sizes of the transcripts are indicated in kilobases.

Table 1

HIV-1 infection of HeLa-P4 (CD4+LTR-lacZ) and derived cell lines stably transfected with CCR5 (HeLa-P5) or US28 (HeLa-P6) expression vectors. Data represent number of blue-stained cells per well (24-well plates after incubation with X-Gal 40 hours after infection).

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Deng et al. (5) did not detect infection with primary HIV-1 strains in a population of CD4+ HeLa cells transduced with a US28 retroviral vector. The extent of US28 expression in these cells was probably lower than that in the HeLa-P6 clone or in transiently transfected cells. Our experiments with epitope-tagged chemokine receptors suggest that the surface expression of US28 is less efficient than that of CCR5. A threshold level of expression necessary to detect HIV coreceptor activity might therefore be more difficult to achieve for US28. Such an explanation also might underlie the apparent lack of HIV coreceptor activity of US28 when expressed with the vaccinia virus–T7 RNA polymerase (vT7pol) system (2); CCR5 and CXCR4 were functional in parallel assays (30). The inhibition of protein synthesis by vaccinia virus infection may affect the transport of US28 or its turnover at the cell surface. Alternatively, it may block the expression of a cellular component required for the coreceptor activity of US28.

Infection by CMV is frequent among HIV-infected individuals and has been proposed to play a role in HIV pathogenesis (31), although this view is not supported by all epidemiological studies (32). The principal HIV-1 target cells, CD4+lymphocytes and monocytes-macrophages, support the replication of CMV in vivo (33). The abundance of CCR5 mRNA in these cells (5, 34) appears markedly lower than that of US28 mRNA in fibroblasts infected with CMV (16). If US28 is expressed at a similar level in CD4+ cells, CMV infection might therefore facilitate subsequent HIV-1 infection, explaining the frequent histological detection of dually infected cells in the brain, lung, or retina (35). In fibroblasts infected experimentally with CMV, US28 was apparently expressed as a late CMV gene (16). If the same pattern of expression occurs in vivo, CMV-infected cells should only express US28 transiently, before they are destroyed by cytopathic effects or by the immune system. However, some cells may survive for a sufficient period to support an HIV replication cycle. Also, US28 has been proposed to be associated with the CMV envelope and to be transferred to the membrane of target cells during virus entry (16). In this instance, cells could bear US28 at their surface before, or even in the absence of, CMV replication.

Several studies have demonstrated up-regulation of HIV-1 replication by CMV infection in CD4+ cell lines (36), macrophages (37), and syncytiotrophoblast cells (38). Various interpretations of these observations have been proposed, but possible effects on HIV-1 entry were not directly tested. However, CMV had no effect or down-regulated HIV-1 replication when cell lines were infected with phenotypically mixed HIV-1 particles via a CD4-independent pathway (39).

The coreceptor activity of CXCR4, CCR5, and US28 was observed for genetically divergent HIV-1 strains and for HIV-2, suggesting that these molecules interact with a conserved domain or conformational motif of gp120. The extracellular domains of these chemokine receptors, which are likely to mediate this interaction, differ from each other in primary structure. In contrast, receptors highly related to CCR5, such as CCR2a, do not mediate HIV-1 entry (5-8). The extracellular domain of US28 might coincidentally possess a conformation similar to that of CCR5 (and CXCR4), allowing interaction with gp120. It is possible that other chemokine receptors also interact with gp120 but lack a property required for HIV coreceptor function, such as the ability to colocalize with CD4. In addition to its possible role in the interactions of CMV and HIV, the US28 chemokine receptor may prove a useful tool with which to address the mechanism of action of HIV coreceptors.

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