Primary SIVsm Isolates Use the CCR5 Coreceptor from Sooty Mangabeys Naturally Infected in West Africa: A Comparison of Coreceptor Usage of Primary SIVsm, HIV-2, and SIVmac

VIROLOGY

246, 113±124 (1998) VY989174

ARTICLE NO.

Primary SIVsm Isolates Use the CCR5 Coreceptor from Sooty Mangabeys Naturally Infected in West Africa: A Comparison of Coreceptor Usage of Primary SIVsm, HIV-2, and SIVmac Zhiwei Chen,* Agegnehu Gettie,* David D. Ho,* and Preston A. Marx*,²,1 *The Aaron Diamond AIDS Research Center, The Rockefeller University, New York, New York 10016; and ²New York University Medical Center, Department of Microbiology, New York, New York 10016 Received January 16, 1998; returned to author for revision February 26, 1998; accepted April 2, 1998 Genetically divergent strains of simian immunodeficiency virus (SIV) from macaques (mac), chimpanzees, and sooty mangabeys (SM) efficiently used rhesus and human CCR5 (R5), but not CXCR4 (xR4), for cell entry. Thus far, however, no studies have characterized primary SIVsm strains for their use of coreceptors derived from their own natural host. Coreceptor usage of two primary, blood-derived SIVsm isolates, SIVsmSL92b and SIVsmFNS from naturally infected sooty mangabeys, was determined. Primary SIVsm efficiently used SM-CCR5 expressed on HOS.CD4 and U87.CD4 cells. Sequence polymorphisms in CCR5 found in four sooty mangabeys did not alter viral entry. Unlike primary rhesus blood-derived R5-tropic SIVmac251, primary SM blood-derived R5-tropic SIVsm was strongly CD4 dependent. The SM-CXCR4 gene was fully functional for xR4-tropic primate lentiviruses, but was not used by primary SIVsm. Therefore, the lack of xR4 tropism among naturally occurring SIVsm strains was not due to CxCR4 gene defects in the natural host. SIVmac derived from four macaques with AIDS also did not use macaque- or SM-derived CXCR4, showing that xR4 tropism did not develop during progression to disease as for humans infected with HIV-1. Three of four primary HIV-2 strains used CCR5 from human, sooty mangabey, and macaque. The fourth, HIV-27924A, obtained from a patient with AIDS, was xR4-tropic. Because SIVmac is most closely related to HIV-2, SIVmac might be expected to mimic tropisms of HIV-2 infections. However, the correlation between xR4 tropism and AIDS may be a species-specific phenomenon limited to humans. The R5-tropic primary SIVsm and HIV-2 strains grew in CCR5-negative human PBMC, consistent with their use of non-CCR5 coreceptors. However, primary SIVsmSL92b did not use non-CCR5 coreceptors efficiently. The two primary SIVsm isolates replicated poorly in CEMx174 cells, which do not express CCR5, compared to CCR5-positive PM1 cells. SIVmac grew equally well in both cell lines. The findings show that SM-chemokine receptors are fully functional for virus entry and that multicoreceptor tropism is a common property of primary lentiviruses within the SIVsm/HIV-2 subfamily. © 1998 Academic Press

lines and macrophages (Cheng-Mayer and Levy, 1988; Cheng-Mayer et al., 1990). Therefore, it was proposed over a decade ago that HIV-1 requires a cell-surface cofactor or coreceptor other than CD4 for efficient entry into target cells. Such coreceptors were recently identified on CD4-bearing cells (Alkhatib et al., 1996; Choe et al., 1996; Deng et al., 1996; Doranz et al., 1996; Dragic et al., 1996). The HIV and simian immunodeficiency virus (SIV) coreceptors belong to the chemokine receptor family, which consists of seven transmembrane-domain G-protein-coupled receptors. Coreceptor usage varies with macrophage-tropic (M-tropic) isolates of HIV-1 using CCR5 (R5), a receptor for the CC chemokines RANTES, MIP-1a, and MIP-1b, and the T-cellline-adapted (TCLA) viruses using CXCR4 (xR4), a receptor for the CXC chemokine SDF-1. These chemokines inhibit HIV-1 infection by blocking entry (Bleul et al., 1996; Cocchi et al., 1995; Oberlin et al., 1996). However, unlike HIV-1, most SIV isolates studied were R5-tropic but not xR4-tropic for entry, regardless of their cell tropism (Chen et al., 1997; Edinger et al., 1997a). Chemokine receptors such as CCR2, CCR3, BOB (also known as GPR15), Bonzo (or called STRL33), GPR1, and US28 also mediate entry of some SIV

INTRODUCTION Shortly after the discovery of human immunodeficiency virus type 1 (HIV-1), the CD4 molecule was recognized as the primary receptor for infection (Dalgleish et al., 1984; Klatzmann et al., 1984; McDougal et al., 1986a). HIV infection is initiated by a fusion step at the cell surface that results from the interaction of HIV envelope glycoproteins and CD4 molecules (Maddon et al., 1986; McDougal et al., 1986a). However, human CD4 molecules, when expressed on nonhuman cell lines, do not render the cells susceptible to HIV-1 infection (Ashorn et al., 1990; Clapham et al., 1991; Maddon et al., 1986). Moreover, this species restriction occurs at the level of viral entry (Broder et al., 1993; Dragic and Alizon, 1993; Dragic et al., 1992), and the entry step probably determines the distinct tropism of HIV on human T-cell

Sequence data from this article have been deposited with the EMBL/ GenBank Data Libraries under Accession No. AF051902±AF051906. 1 To whom correspondence and reprint requests should be addressed at Aaron Diamond AIDS Research Center, 455 First Avenue, 7th Floor, New York, NY 10016. Fax: (212) 725-1126. E-mail: PMarx@ adarc.org. 113

0042-6822/98 $25.00 Copyright © 1998 by Academic Press All rights of reproduction in any form reserved.

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or HIV-1 isolates (Choe et al., 1996; Deng et al., 1997; Doranz et al., 1996; Farzan et al., 1997; Liao et al., 1997). Sooty mangabeys (SM; Cercocebus torquatus atys) are the natural hosts of lentiviruses within the SIVsm/ HIV-2 lineage (Chen et al., 1996; Gao et al., 1994; Hirsch et al., 1989). Thus far, all SIVsm strains tested were R5-tropic, whereas some TCLA HIV-2 strains were xR4tropic in a CD4-independent way (Endres et al., 1996; Reeves and Schulz, 1997). However, no studies have been done with true primary SIVsm strains that were passaged only in sooty mangabey PBMC. To understand the role of coreceptors on viral transmission and pathogenesis, we characterized the CCR5 and CXCR4 genes derived from sooty mangabeys and further compared the coreceptor usage of primary SIVsm, HIV-2, and SIV macaque (SIVmac) strains. SIVmac strains were isolated from rhesus monkeys with AIDS. We found that (1) CCR5 and CXCR4 derived from wild sooty mangabeys facilitate SIV and HIV infection; (2) sequence polymorphisms found in mangabey CCR5 genes do not alter coreceptor function; (3) CCR5 is a major coreceptor for primary SIVsm; (4) primary SIVsm and some primary HIV-2 strains share similarities in coreceptor usage, cell tropism and CD4 dependence for productive infection; and (5) xR4 tropism is not required for AIDS development in SIVmac-infected macaques. RESULTS Sooty mangabey CCR5 and CXCR4 are closely related to their respective rhesus and human genes. To understand the role of the coreceptors CCR5 and CXCR4 in sooty mangabeys, the natural hosts of the SIVsm/ HIV-2 subfamily, CCR5 genes were cloned from cellular DNA of four sooty mangabeys who were free-ranging in Sierra Leone and naturally infected with SIVsm (Chen et al., 1995, 1996). For analysis of the CXCR4 gene, cDNA was prepared from PBMCs of an uninfected mangabey housed at the Yerkes Regional Primate Research Center. Nucleotide and deduced amino acid sequences were determined for the complete coding regions of the cloned genes. Compared to the Hu-CCR5 protein sequence, SM-CCR5 genes were intact in length without the 32-bp deletion previously identified in some humans (Liu et al., 1996). However, amino acid substitutions N13D and V130I were found to be highly conserved in all three nonhuman primate species: macaques, sooty mangabeys, and chimpanzees (Fig. 1A). The four SMCCR5 proteins shared nine amino acid substitutions: I9T, N13D, M49I, I52V, F78L, V130I, K171R, S180P, and P332S. Substitutions S180P and P332S were unique in all four sooty mangabeys, whereas the other seven substitutions were shared with rhesus monkeys. In addition, unique amino acid changes were found at D2E and V25G for SM087; Y3D, M100K, V134G, and T340I for SM079; L107V and V146L for SM089; and V146L for SM085.

Amino acid sequences deduced for a sooty mangabey CXCR4 differed by nine positions from the human protein (Fig. 1A). Five of the nine changes in the mangabey sequence were shared with the CXCR4 of macaques, including 24I, 35H, 38R, 146K, and 176S. Among another four sites, two (32K, 148K) were shared with chimpanzee CXCR4 and two (171G and 184F) were unique for the mangabey studied. As we previously showed, Rh-CCR5 and Rh-CXCR4 facilitated HIV-1 infection (Chen et al., 1997). The amino acid differences shared between sooty mangabey and macaque, therefore, would not cause the loss of their coreceptor function. However, the additional amino acid substitutions could conceivably play a role in the interactions with viral-surface proteins. A phylogenetic analysis using a neighbor-joining method (Saitou and Nei, 1987) showed that SM-CCR5 and SM-CXCR4 are relatively closer to their rhesus than to their human or chimpanzee counterparts (data not shown). They share about 97±99% amino acid identity with their human and rhesus counterparts. Sooty mangabey CCR5 supports the entry of pseudotyped viruses bearing SIVmac and HIV-1 Envs and also the replication of diverse SIV strains. To determine if SM-CCR5 and SM-CXCR4 are active coreceptors, we performed two assays as described previously (Chen et al., 1997). First, the ability of SM-CCR5 to mediate entry of SIVmac and NSI HIV-1 luciferase pseudoviruses was tested. A similar amount of each luciferase reporter virus was used to infect HOS.CD4 and U87.CD4 cells stably expressing SM-CCR5 and SM-CXCR4 derived from five animals. Pseudotypes bearing SIVmac and M-tropic HIV-1 Env efficiently infected cells expressing SM-CCR5 (Fig. 2). In contrast, except for the T-tropic HIV-1 pseudotype, no SIV or M-tropic HIV-1 pseudotypes infected cells expressing SM-CXCR4. Controls confirmed the appropriate specificity of the cells and viruses used. Thus, SMCCR5 and SM-CXCR4 were active for SIVmac and HIV-1, with a specificity similar to that of the analogous human and rhesus coreceptors (Chen et al., 1997). Each SMCCR5 tested contained unique amino acid substitutions, but the sequence polymorphism did not change their coreceptor function. SM-CCR5 was further tested to determine if it supported replication by diverse SIV strains adapted to growth in a human cell line. Coreceptor-expressing HOS.CD4 and U87.CD4 cells were exposed to various strains of SIV. All the SIV strains, including genetically divergent SIVsm, replicated efficiently on HOS.CD4 and U87.CD4 cells expressing CCR5 derived from the wild-caught sooty mangabeys (Table 1). The viruses did not discriminate among human, rhesus, or mangabey coreceptors. Moreover, no SIVsm replicated in cells expressing SM-CXCR4. Thus, the SIV Envs tested were highly specific for CCR5 and did not use CXCR4 to a significant extent, even from the natural species. These data demonstrate that SM-CCR5 but not SM-CXCR4 functions as a coreceptor for the simian lentiviruses tested.

FIG. 1. Amino acid sequence alignment of CCR5 (A) and CXCR4 (B) derived from sooty mangabeys are compared with human, macaque, and chimpanzee proteins. Sequences in boldface were deposited in GenBank. Nucleotide sequences of CCR5 and CXCR4 were obtained from genomic DNA or cDNAs of five sooty mangabeys: SM-079, SM-085, SM-087, SM-089, and SM-Pjg. The seven transmembrane domains (TM) are indicated by lines; amino acid differences with the human sequences are shown in uppercase letters.

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FIG. 2. Entry of luciferase reporter viruses into cells expressing rhesus and sooty mangabey coreceptors. (A) HOS.CD4 cells stably expressing rhesus and sooty mangabey CCR5 or CXCR4 were infected with HIV-1NL4-3 or SIVmac239 luciferase reporter viruses pseudotyped by various SIV (mac1A11 and mac1A11rev), HIV-1 (JR-FL, ADA, and HXB2), or A-MuLV Envs (indicated in parentheses). Mac1A11rev contains the Rev coding sequence in addition to Env. Luciferase activity was measured 4 days after inoculation. HOS.CD4 cells containing pBabe-puro (vector) were used to control for expression of endogenous coreceptors. (B) U87.CD4 cells stably expressing SM-CCR5 or SM-CXCR4 were infected with the reporter viruses as in A. HOS.CD4 cells supported some entry of T-tropic pseudotypes, possibly because of expression of endogenous CXCR4 or some other unidentified coreceptor. When this experiment was repeated, similar results occurred.

Coreceptor usage of primary SIVsm strains. In all previous studies, SIV strains were either passaged in macaque species or adapted in human cell lines (Chen et al., 1997; Edinger et al., 1997a; Marcon et al., 1997). Therefore, it is not known which chemokine receptors are used by primary SIVsm strains, the ancestor group for HIV-2 (Chen et al., 1996; Gao et al., 1994). To address this question, a primary SIVsm from a household pet sooty mangabey was used. SIVsmSL92b is a highly divergent member of the SIVsm/HIV-2 lineage but is most closely related to HIV-2 subtype E (Chen et al., 1996). Another primary SIVsm strain, SIVsmFNS, was used for comparison and was isolated from a naturally infected sooty mangabey at the Yerkes Regional Primate Center. Like other SIV strains, primary SIVsmSL92b and SIVsmFNS infected HOS.CD4 and U87.CD4 cells stably expressing CCR5 but not CXCR4 from three primate species (Tables 2 and 3). Moreover, human-derived

CCR1, CCR2b, CCR3, and CCR4 did not facilitate the infection of SIVsmSL92b and SIVsmFNS in HOS.CD4 cells (data not shown). Table 3 shows that both primary viruses also replicated in human ccr52/ccr52 PBMC, which contain the delta 32-bp homozygous defect. This replication was consistent with the findings that both primary viruses established productive infections in GHOS cells expressing human Bonzo and BOB. In parallel, SIVsm strains grown in human cells, SIVsmSL92a and SIVsmLib-1, also used BOB and Bonzo as a coreceptor (Table 1). Thus, the multicoreceptor tropism is a common property of the genetically divergent SIVsm strains tested. To determine whether primary viruses would replicate in human cell lines, cell-free primary SIVsmSL92b was used to infect CEMx174 and PM1 cells. By FACS analysis using 2D7 and 12G5 monoclonal antibodies, CEMx174 cells did not express CCR5, whereas more

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TABLE 1 Coreceptor Usage of Primate Lentiviruses in HOS.CD4 or GHOS.CD4 Cells Expressing Various Chemokine Receptors HOS.CD4 Human

Sooty Mangabey

Macaque

CCR5

CXCR4

BOBa

Bonzoa

CCR5

CXCR4

CCR5

CXCR4

TCLA SIVsmb SL92a Lib-1

11 11

2 2

11 11

1 11

11 11

2 2

11 11

2 2

SIVmac 239(nef open)/5593 251 316EM

11 11 11

2 2 2

11 11 11

1 1 1

11 11 11

2 2 2

11 11 11

2 2 2

HIV-1 JR-FL

11

2

2

2

11

2

11

2

Virus

Note. 2, no virus growth; 11, p27 . 2 ng/ml; and 1, 0.1 , p27 , 2 ng/ml at the peak; virus growth determined at days 4, 7, and 10 postinfection. a Expressed in GHOS.CD4 cells. b Stocks replicated in CEMx174 cells and MT-2 cells.

than 95% of PM1 cells expressed CCR5 (data not shown). Both cell lines expressed high-level CXCR4 (more than 92%). With an equal virus input (p27 5 10 ng) in CEMx174 cells, SIVmac239 and SIVmac251 replicated robustly within 2 days of infection, while no detectable amount of p27 was found in SIVsmSL92b cultures up to 10 days postinoculation (Fig. 3). In contrast, PM1 cells supported the productive replica tion of the SIVsmSL92b. It is likely that the low infectivity of SIVsmSL92b on CEMx174 is due to the failure

of the virus to efficiently use BOB or other coreceptors on CEMx174 cells. When the SIVsmSL92b input was increased to 20 ng, the p27 antigen was detectable by p27 assay 7 days after infection. These findings show that SIVsmSL92b uses CCR5 on PM1 cells more efficiently than the coreceptors on CEMx174 cells. A similar viral replication kinetic was observed with SIVsmFNS (Fig. 3). Coreceptor usage of four primary HIV-2 isolates. It has been proposed that HIV-2 originated from SIV naturally

TABLE 2 Coreceptor Usage of Primary SIVsm and HIV-2 as Well as SIVmac from Macaques with AIDS in HOS.CD4 or GHOS.CD4 Cells Expressing Various Chemokine Receptors Human

Sooty Mangabey

Macaque

Virus

CCR5

CXCR4

BOBa

Bonzoa

CCR5

CXCR4

CCR5

CXCR4

Primary SIVsm SL92b FNS

11 11

2 2

11 11

1 11

11 11

2 2

11 11

2 2

Primary HIV-2 A195811 A227011 G0415k

11 11 11

2 2 2

11 11 11

11 11 1

11 11 11

2 2 2

11 11 11

2 2 2

SIVmacb 251/1390 251/1434 239/5685 239/5501

11 11 11 11

2 2 2 2

11 11 11 11

1 1 1 1

11 11 11 11

2 2 2 2

11 11 11 11

2 2 2 2

Note. 2, no virus growth; 11, p27 . 2 ng/ml; and 1, 0.1 , p27 , 2 ng/ml at the peak; virus growth was determined at days 4, 7, and 10 postinfection. a Expressed in GHOS.CD4 cells. b SIVmac strains were isolated from macaques at their end stage of AIDS.

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Coreceptor Usage of Primary SIVsm and HIV-2 and SIVmac Strains in U87.CD4 Cells or PBMC Expressing Various Chemokine Receptors U87.CD4 Human

PBMC

Sooty Mangabey CXCR4

CCR5

CXCR4

Macaque

Human ccr52/ccr52

Wild type

2 2

11 11

11 11

11 11 11 2

2 2 2 11

11 11 11 11

11 11 11 11

11 11 11 11

2 2 2 2

11 11 11 11

11 11 11 11

Virus

CCR5

CCR5

Primary SIVsm SL92b FNS

11 11

2 2

11 11

2 2

11 11

Primary HIV-2 A195811 A227011 G0415k 7924A

11 11 11 2

2 2 2 11

11 11 11 2

2 2 2 11

SIVmaca 251/1390 251/1434 239/5685 239/5501

11 11 11 11

2 2 2 2

11 11 11 11

2 2 2 2

CXCR4

Note. Labels are the same as in Table 2. a SIVmac strains were isolated from macaques at the end stage of AIDS.

harbored by sooty mangabeys through cross-species transmission (Chen et al., 1996; Gao et al., 1994). If true, identification of HIV-2 coreceptor usage may help in understanding viral phenotypic variations and adaptations. Four primary HIV-2 strains were tested. All four strains were isolated from West Africans in the similar time period that we isolated SIVsm and in the same geographic regions (Chen et al., 1996; Gao et al., 1994). It was shown that three HIV-2 isolates (A195811, A227011, and G0415k) from asymptomatic individuals used CCR5, replicated in human PBMC with a delta 32-bp homozygous deletion and infected cells expressing BOB or Bonzo (Tables 2 and 3). Because these results are similar to those we found with SIVsm, it is likely that the coreceptor tropism of SIVsm-like viruses remained the same after being transmitted into humans. However, one HIV-2 strain, 7924A, was only xR4-tropic. This virus was the only isolate obtained from a West African with AIDS, which suggests that the R5 HIV-2 may switch to CXCR4 in vivo, a phenomenon that has been observed in some HIV-1-infected individuals (Connor et al., 1997). This finding contrasts with those from SIVsm in that none were xR4-tropic. HIV-27924A coreceptor usage was not characterized on BOB and Bonzo, because the virus infected the parental HOS.CD4 cells, which would interfere with data interpretation. Primary SIVsm strains use CCR5 in a CD4-dependent pathway for productive infection. Some HIV-2 and neurotropic SIV strains have been shown to use CXCR4 and CCR5 in a CD4-independent fashion (Reeves and Schulz, 1997; Edinger et al., 1997b). To test for CD4independent viral entry by primary SIVsm and primary

HIV-2, a subclone of the HOS cell line that expressed Rh-CCR5 was generated. The expression levels of Rh-CCR5 in this cell line were evaluated by FACS analysis using monoclonal antibody 2D7 for cell staining. More than 70% of the cells expressed CCR5 on their surface, similar to HOS.CD4.Rh-CCR5 cells (data not shown). One pseudovirus complemented with SIVmac1A11-Env could enter HOS.Rh-CCR5, but was more than 10-fold less efficient than in entering HOS.CD4.Rh-CCR5 (Figs. 4B and 4F). As we previously determined, HOS.CD4 cells do not allow SIVmac entry; neither do HOS.CD4.Rh-CXCR4 cells. These findings suggest that mac1A11-Env may mediate CD4-independent entry, but this entry was significantly enhanced by the presence of CD4. In contrast, SIVmac239 and SIVmne Env-based pseudoviruses did not enter HOS.Rh-CCR5 sufficiently for infection. As has previously been found (Deng et al., 1997), GHOS.CD4.HuBOB and GHOS.CD4.Hu-Bonzo supported entry of certain SIVmac-based pseudoviruses (Figs. 4D and 4E). To test whether CCR5 alone is sufficient for primary SIVsm and HIV-2 infection, cell-free viruses were used to infect the HOS.Rh-CCR5 cells. Primary PBMC-derived SIVsm strains could not productively infect the cells in the absence of CD4 (Fig. 5). In contrast, primary brain-derived SIV strains, which are primary with respect to the Rhesus macaque, have been reported to use CCR5 independent of CD4 (Edinger et al., 1997b). Although SIVmac316EM, SIVmac251, and HIV2A195811 grew in HOS.Rh-CCR5, the infection was about 10-fold less efficient than when grown in HOS.CD4-RhCCR5 at 10 days after infection. Thus,

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FIG. 3. Replication of primary SIVsm in PM1 (A) and CEMx174 (B) cells. The amount of p27 contained in the inoculum is indicated for each SIV strain. HIV-1JR-Fl and HIV-1NL4-3 (500 TCID50 each) are included as controls. A portion of the supernatant was collected on days 0, 3, 7, and 10 for p27/p24 antigen measurement. Each point is the average of duplicate measurements. SIVsmSL92b (10 ng), SIVsmFNS (10 ng), and HIV-1JR-FL were all negative for growth in CEMx174 cells (B).

most of the SIV and HIV-2 strains tested, including primary SIVsm strains, required CD4 for productive infection. Interestingly, both SIVmac316EM and SIVmac251 were macrophage competent. As it has been shown that macrophage competent SIV uses CCR5 in a way that differs from non-M-tropic SIVmac239 (Edinger et al., 1997a), the CD4-independent usage of CCR5 might be determined by SIVmac Envs. Use of CCR5 but not CXCR4 by SIVmac strains isolated from macaques with AIDS. To test whether a CXCR4 phenotypic switch is required during AIDS development in macaques, four SIVmac strains were isolated from macaques at the end stages of AIDS. Two animals, Rh1390 and Rh1434, had been intravaginally challenged with a parental stock of SIVmac251 and developed AIDS after inoculation (Marx et al., 1996). The new isolates, SIVmac251/1390 and SIVmac251/1434, were obtained from PBMC of these animals collected at the time of euthanasia with AIDS. Two other macaques, Rh5685 and Rh5501, were intravaginally challenged with a parental stock of SIVmac239(nef open)/5593 in a SIVmac-transmission study (Sodora et al., 1998). Similarly, we isolated

SIVmac239/5685 and SIVmac239/5501 from PBMC of these macaques collected before necropsy. Like their parental strains, all four isolates used human or simian CCR5 (Tables 2 and 3) but not human or simian CXCR4. Therefore, it is likely that CXCR4 is not required for AIDS development in the SIV macaque model. Moreover, when we tested the effect of these four isolates on HOS.RhCCR5 cells using the p27 assay, viral infection was not detected, suggesting that all four viral strains use CCR5 in a CD4-dependent way (Fig. 5). Because parental SIVmac251 infected HOS.Rh-CCR5 cells, the failure of SIVmac251/1390 and SIVmac251/1434 to infect the same cells indicates a phenotypic change after intravaginal transmission. Finally, as with their parental viruses, all SIVmac239- and SIVmac251-derived strains strongly used BOB for productive infection. In contrast, replication as measured by p27 was low in Bonzo-expressing cells. Therefore, the findings suggest that CCR5 and BOB are major coreceptors for SIVmac. SIVmac used these coreceptors from the time of virus transmission and did not change coreceptor usage during progression to disease for 1 to 2 years.

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FIG. 4. Entry of luciferase reporter viruses into seven cell lines. The activity of SIVmac luciferase reporter viruses pseudotyped by various SIV (mac1A11, mac239 and mne) or A-MuLV Envs (indicated in parentheses) was measured 4 days after inoculation into (A) HOS, (B) HOS.Rh-CCR5, (C) HOS.CD4, (D) GHOS.CD4.Hu-BOB, (E) GHOS.CD4.Hu-Bonzo, (F) HOS.CD4.Rh-CCR5, and (G) HOS.CD4.Rh-CXCR4 cells.

DISCUSSION We and others previously showed that lentiviruses isolated from sooty mangabeys (SIVsm) use human and rhesus CCR5 as a coreceptor for infection (Chen et al., 1997). However, the role of CCR5 and its polymorphism on SIVsm in its natural hosts was not studied and the coreceptor usage of primary SIVsm strains remained unclear. To better understand the function of CCR5 genes of sooty mangabeys, we focused on four wild animals that were originally found among a group of free-ranging nonhuman primates in Sierra Leone in 1992. Importantly, all four animals were naturally infected with SIVsm, which consisted of a group of genetically divergent viruses within the SIVsm/HIV-2 family (Chen et al., 1996). We now demonstrate that CCR5 and CXCR4 derived from these sooty mangabeys are highly homologous (97±99%) with those from human and macaque. Both SM-CCR5 and SM-CXCR4 function as coreceptors for primate lentiviruses, and two primary SIVsm strains use CCR5, BOB, and Bonzo for infection. SM-CCR5 functions as a coreceptor for SIVsm, including the two primary isolates tested. When the SM-CCR5 genes of different feral mangabeys were compared with human counterparts, several amino acid substitutions were identified. Because a single amino acid substitution completely blocked the coreceptor function for Mtropic HIV-1 in a CCR5 gene derived from an African green monkey (Kuhmann et al., 1997), it was important to know if the changes in the SM-CCR5 genes have a similar effect. We found that all four SM-CCR5 proteins are active in vitro. They facilitate the entry or infection of SIVsm, SIVmac, HIV-2, and M-tropic HIV-1 strains, including SIVsmSL92a, which was isolated from one of four wild mangabeys (Chen et al., 1995). As the viral phenotype may differ between primary and TCLA viruses, we further tested the coreceptor usage of two primary SIVsm

strains. Both viruses used SM-CCR5 but not SM-CXCR4. That CCR5 is used by primary SIVsm strains lends further support for the central role of this chemokine receptor in SIVsm-infected natural hosts. CCR5 is likely a major coreceptor for the natural transmission of SIVsm in sooty mangabeys. In this study, primary SIVsm strains readily infected PM1 cells, which express CCR5, but not CEMx174 cells, which do not. Compared with SIVmac strains, primary SIVsm appeared to require adaptation in order to grow on CEMx174 cells. This adaptation process may be due to the use of coreceptors other than CCR5 on CEMx174 cells. This possibility is supported by the following: (1) when virus input was increased, both primary SIVsm strains replicated with delayed kinetics in CEMx174 cells in the absence of CCR5; and (2) both primary SIVsm strains used BOB, which is a new coreceptor identified recently on CEMx174 cells (Deng et al., 1997). Hence, although BOB may serve as a coreceptor after viral adaptation, CCR5 appears to be a more efficient coreceptor for primary SIVsm transmission. SM-CXCR4 from sooty mangabeys also functions as a viral coreceptor for HIV-1. Because SIVsm-derived viruses do not use CXCR4 as a coreceptor, it may be that the sooty mangabey, the natural viral host, may have defects in its CXCR4 gene; the virus, therefore, could not switch to use CXCR4 during viral transmission and evolution. To test this possibility, we amplified by RT-PCR a CXCR4 gene from the PBMC of an uninfected sooty mangabey and found it contained 11 amino acid substitutions compared to its human counterpart. These substitutions, however, did not change its function as a viral coreceptor. U87.CD4 cells, which do not express endogenous CXCR4, supported infection by both CXCR4-specific HIV-1 and HIV-2 but not by a panel of SIVsm/mac or three primary HIV-2 strains tested. Therefore, the failure

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FIG. 5. Replication of primary SIVsm and HIV-2 strains on (A) HOS.Rh-CCR5 and (B) HOS.CD4.Rh-CCR5 cells. The virus input was 5±10 ng/well of p27 for SIVsm and SIVmac strains and 500 TCID50 for primary HIV-2 strains. A portion of the supernatant was collected on days 0, 4, 7, and 10 for p27 antigen measurement.

of SIVsm to use CXCR4 is probably not due to an intrinsic CXCR4 gene defect in sooty mangabeys. Moreover, our results suggest that SM-CCR5 and SM-CXCR4 do not restrict HIV infection of sooty mangabeys. The block of HIV-1 infection in mangabey cells must be postentry. The similar coreceptor tropism between primary HIV-2 and SIVsm suggests their common origin. Phylogenetic analysis revealed that HIV-2 might originate from SIV naturally harbored by sooty mangabeys through crossspecies transmission events. Because HIV-2 isolates group tightly with SIVsm within the primate lentiviral family (Chen et al., 1996; Gao et al., 1994), sooty mangabeys are most probably the only natural hosts carrying the HIV-2-like lentiviruses. However, HIV-2 isolates have been shown to use CCR5, CXCR4, or both (Endres et al., 1996; Lu et al., 1997; Pleskoff et al., 1997). Moreover, some HIV-2 strains efficiently use CXCR4 for virus entry in a CD4-independent fashion (Endres et al., 1996; Reeves and Schulz, 1997). To further understand the phenotypic similarity between primary SIVsm and primary HIV-2, four primary HIV-2 strains were tested on

stable cell lines expressing different coreceptors. Interestingly, like primary SIVsm strains, three of the four primary HIV-2 strains shared the same coreceptor use on CCR5 or BOB. This result indicates that the coreceptor usage of primary HIV-2 obtained from asymptomatic individuals is similar to that of primary SIVsm and that the viruses therefore retain their ability to use several coreceptors after adaptation to humans. This property also holds for SIVmac isolates obtained from Asian macaques, as SIVmac used both CCR5 and BOB. Unexpectedly, the fourth primary HIV-2 strain, 7924A, used CXCR4 but not CCR5 as a coreceptor. Because 7924A was the only isolate obtained from an AIDS patient, the results suggest that the CXCR4 tropism of HIV-2 might not result from the adaptation of the virus in human T-cell lines in vitro but rather from AIDS development in humans. Our findings agree with a recent study showing that two primary HIV-2 isolates from AIDS patients use CXCR4 (Sol et al., 1997). CXCR4 usage was not required for AIDS development in SIVmac-infected macaques. In some infected

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humans, HIV-1 tropism changes with disease progression (Zhu et al., 1993). During the early phase of infection, virus isolates are typically M-tropic, whereas they tend to be T-tropic late in the disease. It has been hypothesized that this phenomenon, the so-called ``phenotypic switch,'' results from an expansion of coreceptor usage from CCR5 to CXCR4 or other chemokine receptors (Connor et al., 1997). These findings together with our demonstration that HIV-27924A uses CXCR4 suggest that an analogous coreceptor switch or expansion may also exist in simian AIDS models. Therefore, the coreceptor usage of four SIVmac strains isolated from macaques with AIDS was further tested. The coreceptor tropism of two separate challenge stocks, SIVmac239(nef open)/5593 and SIVmac251, was characterized. Previous studies showed that these two viruses use CCR5 but not CXCR4. We now find that these two viruses also use BOB. Interestingly, all four SIVmac strains derived from macaques with AIDS continued to use CCR5 and BOB but not CXCR4. Hence, CXCR4 usage is not strictly required for AIDS development in SIV-infected macaques. The genotypic variation of SIVmac239 after intravaginal challenge was previously characterized. Notably, 120 days after infection, both SIVmac239/5685 and SIVmac239/5501 exhibited distinct genotypes in comparison to their parental stock, SIVmac239(nef open)/5593 (Sodora et al., 1998). These genotypic changes, however, did not alter coreceptor use. This result suggests that the genotypic variation during disease progression may not necessarily affect coreceptor tropism in macaques with AIDS. MATERIALS AND METHODS Virus stocks. The preparation of virus stocks has been previously described (Chen et al., 1997). The virus stocks include (i) SIVmac239/316EM; (ii) SIVmac239(nef open)/ 5593; (iii) SIVmac251; (iv) SIVsmSL92a; and (v) SIVsmLib-1. M-tropic (JR-FL) and T-tropic (NL4-3) HIV-1 strains were used to demonstrate appropriate coreceptor expression on each cell type. Four primary HIV-2 strains (kindly provided by Dr. Beatrice Hahn) (Gao et al., 1994) were originally isolated from West Africans and were propagated and titered in human PBMC in our laboratory. Four SIVmac strains, SIVmac239/5685, SIVmac239/ 5501, SIVmac251/1390, and SIVmac251/1434, were obtained from macaques that developed AIDS (Marx et al., 1996; Sodora et al., 1998). These viruses were isolated and propagated in rhesus PBMC. Luciferase reporter viruses were prepared by cotransfecting 293T cells with pNL-Luc-Env2 or pSIV-Luc-R2E2 and vectors expressing different SIV or HIV-1 Envs, as previously described (Deng et al., 1996).

Cells. Sooty mangabey, rhesus monkey, and human PBMC were prepared by Ficoll gradient separation, as previously described (Chen et al., 1995). Human osteosarcoma HOS.CD4 and glioma U87.CD4 cells, which stably express primate chemokine receptors and CD4, have been described previously (Chen et al., 1997). GHOS.CD4 cells are HOS.CD4-derived cells expressing green fluorescence protein. GHOS.CD4.Hu-BOB and GHOS.CD4.Hu-Bonzo were kindly provided by Dr. Dan Littman at New York University Medical Center. Generation of CEMx174, a T-B-cell hybrid cell line, and PM1, a transformed T-cell line, have also been previously described (Lusso et al., 1995). Isolation of primary SIVsm. A primary SIVsm, SIVsmSL92b, was isolated from a household pet sooty mangabey in Sierra Leone (Chen et al., 1996). To prepare stocks of primary viruses, uninfected sooty mangabey PBMC were obtained from the Yerkes Regional Primate Research Center (Atlanta, GA) and used for isolation and propagation of the virus from the PBMC of the original mangabey host. Therefore, primary SIVsmSL92b was never passaged in another animal species nor in human cell lines. The donor mangabey's PBMC were cultured in interleukin-2 and ConA-containing growth medium. Forty-eight hours after stimulation, 106 donor PBMC were mixed with an equal amount of PBMC from the naturally infected mangabey. The viral replication was measured by a commercial p27 kit (Cellular Products, Buffalo, NY). A second primary isolate, SIVsmFNS, was kindly given by Dr. Frank Novembre (Yerkes Regional Primate Center). SIVsmFNS was originally isolated from a captive sooty mangabey (FNS) at that Center and then propagated in sooty mangabey PBMC as described above. Plasmid construction and expression of SM-CCR5 and SM-CXCR4. The procedure for constructing expression plasmids has been previously described for rhesus CCR5 and CXCR4 (Chen et al., 1997). Briefly, virus stocks for the retroviral vectors expressing CCR5 and CXCR4, pBabe/CCR5 and pBabe/CXCR4, respectively, were prepared by transfecting 293T cells with the MuLV-gag/pol vectors pSV-psi-MLV-env2 and pcVSV-G. Virus was harvested 48 h after transfection and frozen at 270°C. HOS.CD4 and U87.CD4 were infected with 2 ml of the pBabe virus and were selected 48 h later in culture medium containing 1.0 mg/ml puromycin (Sigma, St. Louis, MO). Puromycin-resistant cells were then used in an HIV-1 and SIV Env-mediated syncytium formation assay and for infection with replication-competent or luciferase reporter viruses. To test viral CD4 dependence on Rh-CCR5, stable HOS.Rh-CCR5 cells were made by infecting HOS cells with the pBabe/Rh-CCR5 virus. Virus entry assay. To test for viral entry, cells (2 3 104 to 3 3 104) were seeded in 24-well dishes in culture medium and infected with luciferase reporter virus (20 ng p24 or p27) in a total volume of 300 ml containing 1 ng/ml polybrene. After incubation overnight, 1 ml of medium

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was replaced in each well. After 3 additional days of culture, 100-ml lysates were prepared and luciferase activity in 20 ml was measured using commercially available reagents (Promega, Madison, WI). Infectivity assays. To determine coreceptor usage of different viruses, HOS.CD4 or U87.CD4 cells (2 3 104) stably expressing human or rhesus coreceptors were seeded in 24-well plates. The next day the cells were infected with 5±10 ng/ml of SIV or 500 TCID50 of HIV-1. After 12 h, input virus was removed by replacing the medium three times. The cells were maintained with low serum (2% fetal calf serum) medium for up to 10 days. Virus production was measured by harvesting aliquots of each supernatant and measuring p27 (Cellular Products) or p24 (Abbot, Chicago, IL) by ELISA. After removing 0.5 ml culture supernatant per well for assay, the same amount of fresh medium was added to the culture. To test viral kinetics in CEMx174 or PM1 cells, 1 3 106 cells were seeded in each of the 24 wells per plate. After 12 h, input virus was removed by replacing the culture medium three times. Viral replication was measured as described above. Fluorescence-activated cell sorting (FACS) analysis. Two monoclonal antibodies, 2D7 for CCR5 and 12G5 for CXCR4, were kindly provided by Drs. Charles Mackay and James Hoxie, respectively. PE-labeled secondary antibodies were obtained from CALTAG Laboratories. PE-anti-CD4 antibodies were from Becton Dickinson. These reagents were used to access the expression of CD4, CCR5, and CXCR4 on various cell lines. The staining procedure was done according to the manufacturer's instructions. Sequence analysis. The sequences of chimpanzee and sooty mangabey CCR5 and CXCR4 were determined using an automated sequencer (ABI Prism 377 DNA sequencer). At least two molecular clones were sequenced from each animal using overlapping primers. The sequences of SM-CCR5 and SM-CXCR4 have been submitted to GenBank. ACKNOWLEDGMENTS We thank D. Littman and V. Kewal Ramani for BOB and Bonzo cell lines; Beatrice Hahn for four primary HIV-2 strains; Frank Novembre for SIVsmFNS; Charles Mackay and James Hoxie for 2D7 and 12G5 antibodies, respectively; Zhanqun Jin, Heng Pan, Christine Russo, Amara Luckay, and Yong Guo for technical assistance; Theresa Secrist for Administrative assistance; and Nathaniel R. Landau and Linqi Zhang for critical reading of the manuscript. This work was funded by grants to P.A.M. from the National Institutes of Health (AI38573-02 and R01 AI41420-01).

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