Two Orphan Seven-Transmembrane Segment Receptors Which Are Expressed in CD4-positive Cells Support Simian Immunodeficiency Virus Infection

Messenger RNA was isolated using the CsCl method and selection on magnetic beads with oligo-dT (Dynabeads; DYNAL, Inc., Lake Success, NY). RNA was obtained from purified human CD4⁺ peripheral blood T cells (gift of Dr. Linda Clayton, Dana-Farber Cancer Institutes, Boston, MA), human alveolar macrophages (gift of Dr. Hal Chapman, Brigham and Women's Hospital, Boston, MA), CEM×174 cells, and U87 neuroglioma cells. RNA was also isolated from phytohemagglutinin-treated, interleukin-2–stimulated PBMCs from a healthy rhesus macaque (New England Regional Primate Research Center, Foxboro, MA). The cDNA libraries from the U87 and CEM×174 cell lines were made by reverse transcription (Superscript; GIBCO BRL, Gaithersburg, MD) using a unidirectional primer supplied by the manufacturer. Size-selected cDNAs were cloned into a BstXI/NotI-digested pcDNA3.1 vector (Invitrogen, Carlsbad, CA). The human alveolar macrophage library was prepared by Invitrogen in pcDNA1. Double-stranded CD4⁺ T cell cDNA was synthesized using a kit from Boehringer Mannheim (Indianapolis, IN).

The expression plasmids for rdc1, ebi2, gpr1, gpr15, and dez were prepared by PCR amplification of a cDNA library made from either CEM×174 or U87 cells, as described above. The amplified fragments were cloned into the pcDNA3 plasmid for expression. Expression plasmids for other chemokine receptors were generously supplied by Drs. Paul Ponath and Walter Newman (LeukoSite, Inc., Cambridge, MA) (v28, CXCR1, CXCR2), Dr. Elliot Kieff (Harvard Medical School, Boston, MA) (ebi1), and Dr. Monica Napolitano (Regina Elena Cancer Institut, Rome, Italy) (ter1).

A previously described env-complementation method (27, 28, 47) was used to produce recombinant HIV-1 viruses which contained the SIV envelope glycoproteins and were capable of encoding chloramphenicol acetyltransferase (CAT) in target cells. Briefly, recombinant virus was incubated with Cf2Th cells transfected with plasmids expressing human CD4 and candidate coreceptors. Cells were harvested and assayed for CAT activity, which was determined by measuring the conversion of chloramphenicol to acetylated forms of chloramphenicol. The SIV_mac239 and SIV_mac316 envelope glycoproteins were expressed from the previously described pSIVΔgpv plasmid (47). The HIV-1 envelope glycoproteins were expressed as previously described (27, 28).

The expression of 7-TMS protein messenger RNA in CEM× 174 and U87 cells and primary human CD4⁺ T lymphocytes and alveolar macrophages was examined by synthesis of cDNA from polyadenylated RNA prepared from these cells, as described above. Primers corresponding to the nucleotide sequences encoding the first and third extracellular loops of the proteins were used for amplification by PCR. The identity of amplified fragments was confirmed by restriction enzyme digestion.

We had previously tested a number of human chemokine receptors (CCR1–CCR5, as well as CXCR4) and found that of these only CCR5 could support entry of an HIV-1 virus pseudotyped with the envelope glycoproteins of a pathogenic, molecularly cloned SIV, SIV_mac239 (47). To identify additional coreceptors which might be used by SIV, we screened cDNA libraries from SIV-infectable cells, CEM×174 and U87, for the expression of mRNA encoding known 7-TMS proteins exhibiting some sequence similarity to chemokine receptors. The cDNAs which were shown to be expressed in either cell line were tested for the ability to support SIV and HIV-1 entry. Recombinant HIV-1 viruses which contained either HIV-1 or SIV envelope glycoproteins and expressed CAT were incubated with Cf2Th canine thymocytes transfected with plasmids expressing human CD4 and the 7-TMS proteins. Table 1 lists the 7-TMS proteins tested, summarizes their expression in CEM×174, U87, and human CD4⁺ T cells, and indicates coreceptor activity for viruses with the SIV_mac239 envelope glycoproteins. Of the 7-TMS proteins tested, only gpr1, gpr15, and CCR5 supported the entry of viruses with the SIV_mac239 envelope glycoproteins. These three 7-TMS proteins also supported the entry of viruses with the macrophage-tropic SIV_mac316 envelope glycoproteins (Fig. 1). The SIV coreceptor activity exhibited by the gpr15 protein was greater than that of CCR5, whereas the coreceptor activity of gpr1 was ∼30% that of CCR5 (Table 2). Most of the viruses with HIV-1 envelope glycoproteins (HXBc2, JR-FL, 89.6) did not infect Cf2Th cells expressing CD4 and gpr15, although the viruses with the M-tropic HIV-1 ADA and YU2 envelope glycoproteins demonstrated a low but reproducible signal in these cells (Fig. 1 and Table 2). Following incubation with the ADA and YU2 viruses, the CAT conversion in the CD4⁺, gpr15⁺ Cf2Th cells was <1% of that seen in the CD4⁺, CCR5⁺ control cells (Table 2). Cf2Th cells expressing CD4 and gpr1 were not infected by viruses containing any of the HIV-1 envelope glycoproteins tested (Table 2).

The expression of gpr15 and gpr1 in different cell types was examined. Since specific reagents to detect these proteins were not available, expression was examined by RNA analysis. A cDNA for gpr15 was readily detected in human CD4⁺ T lymphocytes, in human alveolar macrophages, in activated rhesus macaque PBMCs, and in CEM×174 cells, but not in U87 cells (Table 1, Fig. 2, and data not shown). By contrast, a cDNA for gpr1 could not be detected in primary human T lymphocytes, activated rhesus macaque PBMCs, or CEM×174 cells, but was detected in U87 cells and human alveolar macrophages.

HIV-1, HIV-2, and SIV all use CCR5 as a coreceptor, indicating the importance of this protein in primate immunodeficiency virus pathogenesis (28, 30–33, 47, 48). The use of other chemokine receptors by HIV-1 has been suggested to be important for infection of anatomical compartments such as the brain or for more efficient T cell depletion (39, 45, 46). The identification of gpr1 and gpr15 as additional SIV coreceptors should assist efforts to understand the consequences of the use of coreceptors other than CCR5 in primate models of AIDS. While the in vivo contribution of gpr15 to SIV replication and pathogenesis requires further investigation, several lines of evidence indicate that gpr15 is an important SIV coreceptor. The gpr15 protein is expressed on CD4⁺ T lymphocytes, a major target cell for SIV infection in vivo (10), and on alveolar macrophages. The gpr15 protein is also expressed on CEM× 174 cells, which are routinely used to passage SIV obtained from monkey PBMCs (52). The rapid outgrowth of SIV viruses with minimal sequence changes on CEM×174 cells suggests that these cells express a receptor used by primary SIV viruses. The weak use of gpr15 by the ADA and YU2 HIV-1 viruses may be an inadvertent consequence of similarities in the amino-terminal regions of gpr15 and CCR5, or may indicate that adaptation to these receptors or to a related receptor occurs in some subsets of HIV-1.

The in vivo contribution of gpr1 to primate immunodeficiency virus infection is also unresolved. The gpr1 protein weakly supported SIV infection in our studies. Whether this inefficient coreceptor activity is an intrinsic property of gpr1 or merely reflects low cell surface expression of gpr1 requires further investigation. While gpr1 is not apparently expressed on primary CD4⁺ lymphocytes, it is expressed on tissue macrophages and in the brain (53) and thus may play a role in SIV infection of particular nonlymphoid target cells.

In primary structure, gpr1 and gpr15 resemble the angiotensin II receptor and the orphan receptors dez and apj more than they do any of the known chemokine receptors (53, 54). Gpr15, like dez and gpr1, lacks the cysteines in the NH₂-terminal region and the third extracellular loop which, in the chemokine receptors, are thought to be disulfide linked. It is interesting that despite the general sequence divergence of gpr15/gpr1 and other identified primate immunodeficiency virus coreceptors the gpr15 and gpr1 amino termini contain three tyrosines which align with similarly positioned tyrosines in CCR5 (Fig. 3). Alteration of these tyrosines has been shown to decrease the efficiency with which CCR5 supports the entry of SIV and M-tropic HIV-1 isolates (Farzan, M., H. Choe, and J. Sodroski, unpublished observations). The identification of gpr15 and gpr1 as SIV coreceptors suggests a greater range and complexity of coreceptors for the primate immunodeficiency viruses than previously described. Comparative studies of these divergent coreceptors with the known coreceptors for these viruses should assist in the identification of common structural elements in 7-TMS proteins which serve as viral entry cofactors.

We thank Dr. Ronald Desrosiers for the gift of the SIV_mac239 and SIV_mac316 infectious proviral clones and Drs. Paul Ponath, Walter Newman, Elliot Kieff, and Monica Napolitano for plasmids expressing chemokine receptors. We thank Ms. Lorraine Rabb and Ms. Yvette McLaughlin for manuscript preparation.

This work was supported by a grant to Joseph Sodroski from the National Institutes of Health (AI-24755) and by a Center for AIDS Research grant to the Dana-Farber Cancer Institute (AI-28691). Dana-Farber Cancer Institute is also the recipient of a Cancer Center grant from the National Institutes of Health (CA-06516). Luisa Marcon was supported by National Cancer Institute National Research Science Award Training Grant (CA-09382) and by an award from Istituto Superiore di Sanitá and by the University of Padua. Craig Gerard was supported by National Institutes of Health grants HL-51366 and AI-36162 as well as by the Rubenstein/Cable Fund at the Perlmutter Laboratory. This work was made possible by gifts from the late William McCarty-Cooper, from the G. Harold and Leila Y. Mathers Charitable Foundation, and from the Friends 10.