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Copyright © 1998, American Society for Microbiology

A Broad Range of Chemokine Receptors Are Used by Primary

Isolates of Human Immunodeficiency Virus Type 2

as Coreceptors with CD4

A

´ INE M

C

KNIGHT,

1

* MATTHIAS T. DITTMAR,

1

JOSE´ MONIZ-PERIERA,

2

KOYA ARIYOSHI,

3

JACQUELINE D. REEVES,

1

SAM HIBBITTS,

1

DENISE WHITBY,

1

EMMA AARONS,

4

AMANDA E. I. PROUDFOOT,

5

HILTON WHITTLE,

3AND

PAUL R. CLAPHAM

1

Section of Virology, Chester Beatty Laboratories, Institute of Cancer Research,

1

and Department of GUM,

St. Mary’s Hospital,

4

London, United Kingdom; Faculdade de Farmacia, Universidada de Lisboa,

Lisbon, Portugal

2

; Medical Research Council Laboratories, Fajara, The Gambia

3

; and Geneva

Biomedical Institute, Glaxo Wellcome Research and Development SA, Geneva, Switzerland

5

Received 29 September 1997/Accepted 21 January 1998

Like human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV), HIV-2

re-quires a coreceptor in addition to CD4 for entry into cells. HIV and SIV coreceptor molecules belong to a family

of seven-transmembrane-domain G-protein-coupled receptors. Here we show that primary HIV-2 isolates can

use a broad range of coreceptor molecules, including CCR1, CCR2b, CCR3, CCR4, CCR5, and CXCR4. Despite

broad coreceptor use, the chemokine ligand SDF-1 substantially blocked HIV-2 infectivity of peripheral blood

mononuclear cells, indicating that its receptor, CXCR4, was the predominant coreceptor for infection of these

cells. However, expression of CXCR4 together with CD4 on some cell types did not confer susceptibility to

infection by all CXCR4-using virus isolates. These data therefore indicate that another factor(s) influences the

ability of HIV-2 to replicate in human cell types that express the appropriate receptors for virus entry.

CD4 is the primary cell surface receptor for entry of human

immunodeficiency virus (HIV) and simian immunodeficiency

virus (SIV) into cells. Coreceptors for HIV and SIV have been

identified as G-protein-coupled receptors with seven

trans-membrane domains (reviewed in references 14, 42, 59, and 60).

Early evidence suggested that there may be more than one

coreceptor, reflecting the extended tropism of

syncytium-in-ducing (SI) HIV-1 strains for CD4-positive T-cell lines

com-pared to non-syncytium-inducing (NSI) strains, which infect

mainly primary macrophage and T-cell cultures. CXCR4 was

the first HIV type 1 (HIV-1) coreceptor to be defined (25) and

was shown to be the major coreceptor for T-cell-line-adapted

and primary SI HIV-1 isolates (21, 25, 40, 54, 61). CCR5 is the

main coreceptor for primary NSI HIV-1 isolates (3, 10, 18, 21,

22, 61). HIV-1 viral entry is inhibited in the presence of the

ligands to these chemokine receptors. Thus, RANTES,

MIP-1

a

, and MIP-1

b

, the ligands for CCR5, inhibit

macrophage-tropic isolates, while SDF-1, the specific ligand for CXCR4,

inhibits entry of T-cell-tropic isolates (5, 15, 34, 44).

CCR5 is important for transmission. Individuals with a

CCR5 gene deletion are largely protected from HIV-1

infec-tion (17, 31, 33, 37, 46, 50). CCR5-using strains are present

throughout the course of infection in an individual, while

CXCR4-using viruses often develop late in disease (16). Some

HIV-1 isolates have the ability to exploit other chemokine

re-ceptor molecules, such as CCR3, CCR2b, BOB, and BONZO

(4, 10, 19, 21, 24, 36), in addition to CCR5 and CXCR4. The

efficiency of these other coreceptors for HIV entry is

contro-versial.

Phylogenetic analysis has shown that HIV-1 clusters with

SIV isolated from chimpanzees (SIVcpz) and that HIV-2

clus-ters with SIV from sooty mangabeys (SIVsm) (7, 26, 27, 43).

HIV is likely to have originated as a result of cross-species

transmission of SIV from African apes or monkeys to humans

(53). Like HIV, SIV also requires a coreceptor in addition to

CD4 for viral entry, since several nonhuman cell types

express-ing human CD4 fail to support SIV viral entry (39). Chen et al.

reported that SIV isolates (SIVmac, SIVsmSL92a,

SIVsmLib-1, and SIVcpz GAB) could use human or rhesus monkey CCR5

for entry but not human CXCR4, CCR1, CCR2b, CCR3, or

CCR4 (8). SIV, however, could infect several cell lines (now

known to be CCR5 negative) that resist HIV-1 infection,

in-dicating that other coreceptors may be exploited by SIV (12,

32). Several groups have recently reported three likely

candi-dates for this unknown receptor(s). BOB/gpr15 (19, 24, 29) and

BONZO/STRL33 (4, 36) both assist entry of SIVmac and

HIV-2 as well as certain SI and NSI strains of HIV-1. Another

orphan receptor, GPR1, allows entry of SIVmac but not HIV-1

into CD4-positive cells (24).

Coreceptors used by the laboratory-adapted isolate HIV-2

ROD were recently studied by Bro¨n et al. (6). In cell-to-cell

fusion assays, ROD was particularly promiscuous and induced

fusion of CD4-positive cells expressing CCR1, CCR2b, CCR3,

CCR5, CXCR2, and CXCR4. In contrast, in cell-free

infectiv-ity assays, ROD used CCR3 and CXCR4 but not CCR5. A

variant of HIV-2, ROD/B, that efficiently infects some cell

types in the absence of CD4 (13) also used CXCR4 and CCR3

or an orphan receptor, V28, in the absence of CD4 (23, 48).

Re-cently, Sol et al. (56) showed that CCR3, CCR5, and CXCR4

were used by HIV-2 primary strains, while Heredia et al. (30)

reported that some primary isolates of HIV-2 could use CCR1,

CCR2b, and CCR5 as well as CXCR4 but not CCR3 or CCR4.

However, there was no indication of the relative efficiencies of

these infections. Deng et al. (19) also showed that some

pri-mary HIV-2 isolates used several coreceptors, including the

newly identified BOB/gpr15 and BONZO/STRL33.

Here we report on the coreceptor use of various primary

* Corresponding author. Mailing address: Section of Virology,

Chester Beatty Laboratories, Institute of Cancer Research, London

SW3 6JB, United Kingdom. Phone: 8133. Fax:

44-171-352-3299. E-mail: aine@icr.ac.uk.

4065

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isolates of HIV-2 isolated mainly from symptomatic patients

and cultured in peripheral blood mononuclear cells (PBMCs).

We find that these primary HIV-2 isolates can use a broad

range of coreceptors, including CCR1, CCR2b, CCR3, CCR4,

CCR5, and CXCR4. Not all human cell types that express an

appropriate coreceptor supported virus replication, indicating

the presence of other factors influencing viral tropism.

MATERIALS AND METHODS

Cells.U87/CD4 cells expressing chemokine receptors CCR1, CCR2b, CCR3, CCR5, and CXCR4 were a gift from Dan Littman and have been previously described (18). CCC/CD4 cells derived from cat kidney (CCC S1L2), RD/CD4 cells derived from human rhabdomyosarcoma, and HeLa/CD4 cells have been previously described (12, 39). SCL/CD4 cells are from human skin (9), and WI-38/CD4 cells are simian virus 40-transformed human lung fibroblasts (28). Human CD4 was stably expressed on these cell types by using an amphotropic retroviral vector (9). MT-2 (41), C8166 (11), Molt 4, H9, and Sup T1 cells are all human CD41T-cell lines and have been described elsewhere (13, 39). Peripheral

blood PBMCs were stimulated for 2 days with phytohemagglutinin (PHA) (0.5 mg/ml) and then cultured in RPMI plus 10% fetal calf serum (FCS) supple-mented with interleukin-2 (IL-2) (20 U/ml).

Chemokines and coreceptor ligands.A recombinant form of SDF-1 that retains the N-terminal methionine residue was used. This, MCP-1, and AOP-RANTES (55) were provided by Glaxo Wellcome. vMIP-II, a chemokine en-coded by the Kaposi’s sarcoma-associated herpesvirus (35), was provided by T. W. Schwartz, University of Copenhagen. AMD3100 is a bicyclam derivative that reacts with CXCR4 (51).

Isolation of virus and preparation of stocks.CBL-20, CBL-23, and V9 were isolated from individuals from The Gambia. CBL-20 and CBL-23 have been described before (52). CBL-20 and V9 were isolated from individuals with AIDS, whereas CBL-23 is from an individual with AIDS-related complex. prCBL-20 was propagated only in primary PBMC cultures, while CBL-20 was adapted for growth in T-cell lines. MIR was isolated from an AIDS patient who died in 1987. A-ND was from a Portuguese individual with progressive disease. ST was from a Senagalese asymptomatic individual (53). Viruses were prepared as follows. PBMCs from HIV-2-infected patients were cocultured with PHA-stimulated uninfected PBMCs in the presence of recombinant IL-2 (20 IU/ml). Supernatant from a day 28 culture was passaged into a culture of fresh uninfected PBMCs, which was monitored weekly for reverse transcriptase (RT) activity. When the culture became positive for RT activity (.50 pg/ml), fresh PBMCs were added and virus was allowed to grow for a further week before the supernatant was harvested. Stocks of ST were made from Molt 4-derived virus passaged into PBMCs. Virus aliquots were stored under liquid N2.

Plasmids.The chemokine receptors CCR1, CCR2b, CCR3, CCR4, CCR5, and CXCR4 were subcloned into the vector pcDNA3.1 (Invitrogen) for trans-fection and transient expression in CCC/CD4 cells.

Transfection of CCC/CD4 cells.Cells were plated overnight in six-well trays at 23105cells per ml. For each well, 1mg of plasmid DNA in 100ml of FCS-free medium was added to a solution of 7ml of Lipofectamine (GIBCO BRL) (1 mg/ml) in 100ml of FCS-free medium and allowed to stand on the bench for 45 min. Cells were washed twice before addition of 0.8 ml of FCS-free medium followed by the plasmid and Lipofectamine mixture. Control transfections (mock) were done by using the vector pcDNA3.1 without insert. After 5 h of incubation, the medium was replaced, and after overnight incubation, the cells were set up in 24-well trays for infection the following day.

Syncytium induction assays.High-titer virus stocks were added to 0.5 ml of target cells (23105cells/ml for MT-2, C8166, Molt 4, and Sup T1 cells or 13 106cells/ml for PBMCs) in a 48-well tray. Cells were passaged twice weekly for 2 weeks and scored for syncytia as follows:2, no syncytia;1, at least one syncytium in cultured cells but fewer than 5% of nuclei in syncytia;11,.5% but ,19% of nuclei in syncytia,111, 20 to 40% of nuclei in syncytia; and1111, .40% of nuclei in syncytia. After 2 weeks, RT activity in the PBMC cultures was measured to assess the presence of virus.

Determination of RT activity. Virus production into the supernatant was assayed by measurement of RT activity by a sensitive nonradioactive method (Retrosys RT activity kit; Cavidi Tech, Uppsala, Sweden).

Virus infectivity and inhibition assays.Cells were plated at 53104per well in a 24-well plate or at 13104per well in a 48-well dish. A total of 0.1 ml (48-well dishes) or 0.2 ml (24-well plates) of 10-fold serial dilutions of virus was added to cells and incubated at 37°C for 1 h before washing in growth medium and reincubation for 3 to 5 days. Infected cells were detected by specific immuno-staining. For titration of virus on PBMCs, virus (50ml) was added to 105cells per well of a 96-well tray. For inhibition-of-infection studies with PBMCs, cells were first treated with SDF-1 at 400 or 1,600 ng/ml, with AOP-RANTES at 200 or 800 ng/ml, or with MCP-1 at 800 ng/ml for 20 min before addition of virus.

Immunostaining of HIV-infected cells.The immunostaining method has been described before (39). Briefly, HIV-infected cells were washed in serum-free phosphate-buffered-saline (PBS) and fixed in methanol-acetone (1:1) at220°C. After washing of the cells in PBS–1% FCS (PBS-FCS), HIV-2-positive serum

was added at 1/5,000 (in PBS-FCS) to detect virus antigen. After three washes in PBS-FCS, the cells were incubated for 1 h with mouse anti-human immunoglob-ulin G conjugated withb-galactosidase (Southern Biotech). After a further three washes in serum-free PBS, 0.5 mg of X-Gal (5-bromo-4-chloro-3-indolyl-b-D

-galactopyranoside) substrate (Novolabs) per ml in PBS containing 3 mM potas-sium ferricyanide, 3 mM potaspotas-sium ferrocyanide, and 1 mM magnepotas-sium chloride was added. Clusters of blue cells were counted as foci of infection (focus-forming units [FFU]) to estimate the virus titer.

PCR amplification of CCR5 genes.Oligonucleotide primers spanning the CCR5 32-bp deletion (D32) were used to amplify DNA fragments from PBMC cultures (37). This procedure produces a PCR DNA fragment of 183 bp from wild-type CCR5 DNA and a fragment of 151 bp of DNA carrying the 32-bp CCR5 deletion. Genomic DNA was derived from PBMCs by using a Nucleon Biosciences (Coatbridge, United Kingdom) kit. The primer pair used was 7591 CTT CAT TAC ACC TGC AGC TCT and 9412ACC AGC CCT GTG CCT CTT CTT. The samples were initially denatured at 95°C for 5 min, followed by 30 cycles of a denaturing step at 95°C for 30 s, an annealing step at 45°C for 30 s, and an extension step at 72°C for 30 s. A final step at 72°C for 5 min was done. PCR products were visualized by electrophoresis on a 3% agarose gel.

RESULTS

Coreceptor use by primary isolates of HIV-2.

We

deter-mined the coreceptor use of six primary isolates of HIV-2

grown in PBMCs. ST came from an asymptomatic Senegalese

individual (53). The remaining isolates were from symptomatic

patients. MIR and A-ND were isolated from two symptomatic

Portuguese patients. Three isolates from Gambian individuals

were also included. Two of these Gambian isolates, V9 and

prCBL-20, were from patients with AIDS, while prCBL-23 was

from an individual with AIDS-related complex. To determine

which coreceptors these viruses used, we challenged U87/CD4

cells stably expressing each of the chemokine receptors, CCR1,

CCR2b, CCR3, CCR5, and CXCR4. Virus isolates MIR, ST,

V9, prCBL-20, and prCBL-23 showed efficient use of all five

chemokine receptor molecules in cell-free infection (Fig. 1).

No infection of the parental U87/CD4 cells was detected. MIR,

prCBL-20, and prCBL-23 plated most efficiently on cells

ex-pressing CXCR4, while ST preferentially infects cells

express-ing CCR5. Interestexpress-ingly, A-ND showed no detectable activity

on CCR5-expressing cells.

To verify the infectivity results gained from the set of U87/

CD4 cells expressing coreceptors, we tested three isolates,

MIR, V9, and prCBL-20, on cat CCC/CD4 cells transfected

with and transiently expressing each of the chemokine

recep-tors tested on U87/CD4 cells. Although this procedure yielded

variable results, we were able to confirm that these viruses

were capable of using a broad range of coreceptors. Table 1

shows a representative set of results from one experiment

using MIR. We also tested whether these isolates used CCR4

(not included in the U87/CD4 experiments) in transiently

transfected CCC/CD4 cells. Although MIR used CCR4

con-sistently, low levels of infectivity were observed with the two

other strains, V9 and prCBL-20.

U87 and CCC cells express undefined coreceptors that can

be used by some HIV-2 strains (12, 39). It was possible that the

chemokine receptor-positive subclones of U87/CD4 may

ex-press such undefined coreceptors at higher levels than parental

U87/CD4 cells and therefore confer infection independently of

the expressed recombinant chemokine receptor. To rule out

this possibility, we tested whether specific coreceptor ligands

could inhibit A-ND infection of U87/CD4 cells expressing

CCR1, CCR3, or CXCR4. vMIP-II is a chemokine encoded

by Kaposi’s sarcoma-associated herpesvirus that

preferential-ly blocks HIV-1 infection via CCR3 (35). vMIP-II (1

m

g/ml)

reduced A-ND infectivity for CCR3

1

U87/CD4 cells by over

90% but had no effect on infection via CCR1 or CXCR4.

Similarly, a 100-ng/ml concentration of AMD3100, a

bicy-clam derivative that binds CXCR4, blocked A-ND infection

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of CXCR4

1

U87/CD4 cells by over 90% but did not inhibit

CCR1- or CCR3-dependent infection (data not shown).

Coreceptor use by T-cell-line-adapted HIV-2 isolates.

One

isolate, prCBL-20, was adapted for replication in a T-cell line

(H9). The virus strain derived, CBL-20 (52), was then tested

for coreceptor use on the U87/CD4 cells expressing different

chemokine receptors. Figure 2 shows that selection of primary

prCBL-20 to grow in T cells resulted in a virus variant which

could infect only CXCR4

1

U87/CD4 cells. Unlike the parental

prCBL-20 virus, CBL-20 had lost the capacity to infect U87/

CD4 cells via CCR1, CCR2b, CCR3, or CCR5. Thus, selection

into T-cell lines resulted in restricted coreceptor use for this

virus.

Phenotypes of HIV-2 strains used.

All of the isolates studied

here could use CXCR4 efficiently as a coreceptor molecule.

For HIV-1, use of CXCR4 generally correlates with an SI

phe-notype (54, 61), whereas all NSI isolates use at least CCR5 (3,

10, 18, 21, 22). We tested whether our isolates could be

cate-gorized as NSI or SI according to the conventional criteria

used for HIV (1, 58). Each isolate was plated on MT-2 cells,

PBMCs, and a number of T-cell lines. Syncytium induction on

these cell types indicates the presence of an SI virus. The

re-sults are shown in Table 2. At least two of these isolates, V9

and prCBL-20, did not induce syncytia in any of the cell types

tested. MIR, A-ND, ST, prCBL-23, and CBL-20, however,

in-duced syncytia in at least one of the T-cell lines tested.

Infection of PBMCs from an individual who is homozygous

for the

D

32 CCR5 gene.

We assessed whether the primary

HIV-2 strains could infect PHA- and IL-2-stimulated PBMCs

derived from an individual who was homozygous for the

D

32

CCR5 gene. Figure 3 shows a smaller PCR-amplified DNA

fragment from PBMCs derived from an individual homozygous

for deletions of CCR5. These cells were resistant to the CCR5

with HIV-1 strain SF-162 (55a). All but one of the primary

HIV-2 strains used CCR5 (see above), while all could use

several other coreceptors, including CXCR4. Table 3 shows

that all strains tested replicated as efficiently in

D

32/

D

32 CCR5

PBMCs as in wild-type CCR5 PBMCs and therefore do not

depend on CCR5 for infection of PBMCs.

[image:3.612.53.547.69.358.2]

Inhibition of infection of primary HIV-2 isolates by the

ligands to specific chemokine coreceptors.

We next

investi-gated the coreceptor molecules used by A-ND, prCBL-20, and

CBL-20 to infect PBMCs in vitro. PBMCs were derived either

from individuals homozygous for

D

32 CCR5 or from wild-type

donors. We tested for infectivity of cells treated with SDF-1

(400 and 1,600 ng/ml), AOP-RANTES (200 and 800 ng/ml),

and MCP-1 (800 ng/ml). AOP-RANTES is an analog of

RAN-TES and a potent inhibitor of HIV-1 infection (55) and is also

a ligand for CCR5, CCR3, and CCR1 (47). Figure 4 shows that

whatever the source of PBMCs, SDF-1, the natural ligand for

FIG. 1. Titration of primary HIV-2 isolates on chemokine receptor-expressing U87/CD4 cells. After incubation for 3 to 5 days, cells were fixed and immunostained and infection (FFU per milliliter) was estimated. Error bars indicate standard deviations.

TABLE 1. HIV-2 MIR infection of CCC/CD4 cells expressing

different chemokine receptors after transfection

Chemokine

receptor Infectivity of HIV-2 MIR(FFU, mean6SD)

Control ...

0

CCR1... 150

6

70

CCR2b ... 440

6

84

CCR3... 530

6

42

CCR4... 440

6

56

CCR5... 990

6

268

CXCR4... 1,050

6

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CXCR4, inhibited the infection of all three viruses by more

than 90% (at 1,600 ng/ml). In contrast, MCP-1, a natural

li-gand for CCR2b and CCR4, had no effect on the infectivity of

PBMCs by prCBL-20. In some experiments AOP-RANTES

showed some inhibition early on when RT activity was first

detected in control infections after infection of wild-type

PB-MCs (data not shown). This inhibition was not sustained.

These results therefore indicate that CXCR4 is the

predomi-nant coreceptor used in vitro by the three HIV-2 strains tested

for PBMC infection.

Tropism of primary HIV-2 isolates for CD4-expressing cell

lines.

We determined the abilities of the HIV-2 isolates to

in-fect various human cell lines transin-fected with and stably

ex-pressing human CD4. Each of these cell types (HeLa/CD4,

RD/CD4, WI-38T/CD4, SCL/CD4, and U87/CD4/CXCR4)

was tested for expression of CCR5 or CXCR4 by using specific

monoclonal antibodies followed by immunostaining and flow

cytometric analysis (data not shown). As reported previously,

HeLa/CD4 and RD/CD4 cells both express CXCR4 to high

levels (40) but do not express CCR5 (data not shown). The

SCL/CD4 or WI-38/CD4 cells did not express either CXCR4

or CCR5 at the cell surface. All seven virus isolates were

titrat-ed on these cell types as well as on U87/CD4/CXCR4 cells

(Ta-ble 4). MIR, A-ND, V9, prCBL-20, and CBL-20 can efficiently

infect HeLa/CD4 or RD/CD4 cells, presumably through CXCR4,

which is expressed on both of these cell types. Interestingly, ST

and prCBL-23, both of which efficiently infect CXCR4

1

U87/

CD4 cells, did not infect either CXCR4

1

HeLa/CD4 or RD/

CD4 cells even when the appropriate coreceptor was

ex-pressed. Thus, there is a further restriction to infection of some

isolates on these cell lines. Furthermore, the ST isolate, unlike

the other isolates tested, infected SCL/CD4 and WI-38/CD4

cells. Since ST, like other strains tested here, can use CCR1 to

-5 and CXCR4, these results suggest that another cell surface

receptor may be expressed on these two cell types.

DISCUSSION

[image:4.612.135.467.67.203.2]

This study determined the abilities of six primary isolates

and one laboratory-adapted isolate of HIV-2 to use six

che-mokine receptors as coreceptor molecules for fusion and

in-fection of human cells expressing CD4. The results presented

here show that, unlike HIV-1 and SIV, the primary isolates of

HIV-2 tested used a wide range of chemokine receptors. Five

of the isolates, MIR, ST, V9, prCBL-20, and prCBL-23, could

use CCR1, CCR2b, CCR3, CCR5, and CXCR4 expressed on

U87/CD4 cells. This broad coreceptor use was confirmed for

MIR, V9, and prCBL-20 on CCC/CD4 cells transiently

trans-fected with these chemokine receptors. One isolate, A-ND,

FIG. 2. Selection of a primary isolate to grow in T-cell lines restricts coreceptor use. prCBL-20 was selected to grow in C8166 cells, and stocks were made in H9 cells (52). The resulting virus was titrated on either the parental U87/CD4 cells or those stably expressing the chemokine receptor CCR1, CCR2b, CCR3, CCR5, or CXCR4. Infection (FFU per milliliter) was calculated after immunostaining of 5-day-old infected cultures. Error bars indicate standard deviations.

[image:4.612.369.486.504.665.2]

FIG. 3. Detection of homozygous deleted CCR5. CCR5 DNA was amplified from genomic DNA derived from the PBMCs of an individual homozygous for the CCR5 deletion and an individual homozygous for the wild-type CCR5 gene. PCR products were visualized by electrophoresis on a 3% agarose gel. Lanes: M, 1-kb marker; 1, negative control; 2 and 3, 200 and 400 ng of homozygousD32 CCR5 PBMC DNA, respectively; 4, negative control; 5 and 6, 200 and 400 ng of wild-type CCR5 PBMC DNA, respectively.

TABLE 2. Phenotype determination of primary HIV-2 isolates

a

Target cell

Syncytium formationbby the following virus:

MIR A-ND ST V9 pCBL20 CBL-20 pCBL23

Molt 4

11

11

1

2

2

111

2

Sup T1

1

2

2

2

2

1

2

C8166

1111 111 11

2

2

111

2

MT-2

1

2

2

2

2

2

1111

PBMC

1

2

2

2

2

2

2

aThe phenotypes (SI or NSI) of the primary isolates and laboratory-adapted

CBL-20 were determined. Viruses were analyzed for their ability to induce syncytia in the cell lines indicated. Cells were infected with virus, passaged twice weekly for 2 weeks, and then scored for syncytia.

b2, no syncytia seen;1, at least one syncytium in cultured cells but fewer than

5% of nuclei in syncytia,11,.5% but,19% of nuclei in syncytia;111, 20 to 40% of nuclei in syncytia;1111,.40% of nuclei in syncytia. Supernatants from PBMCs were tested for RT activity (see Materials and Methods) after 2 weeks to verify the presence of virus; for each virus the RT activity was.500 pg/ml.

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is peculiar in that we could not detect any infectivity on

CCR5-expressing cells yet it used CCR1, CCR2b, CCR3, and

CXCR4. MIR, V9, and prCBL-20 were also tested for

infec-tion of CCC/CD4 cells transfected with and expressing CCR4

(not tested in the U87/CD4 panel). We found that all of these

virus isolates could also use CCR4 at least to some extent. This

is the first report of any HIV or SIV isolates using CCR4.

Recently, Heredia et al. (30) also showed that primary

HIV-2 isolates used several coreceptors. However in contrast to the

viruses we tested, several isolates that used only CCR5 were

identified, as well as some that additionally used CXCR4,

CCR1, and CCR3 expressed by HOS cells. It is therefore

pos-sible that HIV-2 coreceptor use broadens as disease

pro-gresses, as suggested for HIV-1 by Connor et al. (16). Apart

from ST, all of the HIV-2 strains tested here were derived from

symptomatic patients. Our results therefore support the

hy-pothesis of Connor et al.

For transmission of HIV-1, CCR5 is clearly the most

impor-tant coreceptor. The majority of isolates thus far reported use

CCR5 either exclusively (for NSI isolates) or in addition to

CXCR4 (for SI isolates). Early after HIV-1 infection and in

the asymptomatic phase, NSI CCR5-using viruses are usually

isolated (16), suggesting that there is a strong selective

pres-sure for the spread of CCR5-using viruses. As disease

devel-ops, CXCR4-using viruses can be more frequently isolated,

from about 50% of patients with AIDS. How the broad

core-ceptor use by HIV-2 strains shown here influences their

trans-mission remains to be determined.

[image:5.612.49.291.81.186.2]

We tested the coreceptor use of prCBL-20 after its

adap-tation to culture in H9 cells. The isolate derived (CBL-20)

showed a marked loss in its ability to use the broad range of

coreceptors used by the parental virus and could use only

CXCR4 efficiently. Furthermore, T-cell-line-adapted CBL-20

could use CXCR4 expressed on HeLa/CD4 cells, whereas

prCBL-20 could not. In contrast, prCBL-20 but not CBL-20

infected primary macrophages (not shown) even though they

express CXCR4 (40). Recently Bro¨n et al. (6) showed that the

T-cell-line-adapted ROD strain of HIV-2 infected

CXCR4-and CCR3-expressing CD4

1

cells. Interestingly, while cell-free

[image:5.612.102.486.389.688.2]

FIG. 4. Inhibition of infection by prCBL-20, CBL-20, and A-ND with chemokines on wild-type CCR5 andD32 CCR5 PBMCs. Infectivity assays with prCBL-20 and A-ND were performed in the absence and presence of the chemokines SDF-1, AOP-RANTES, and MCP-1 either alone or together at the concentrations indicated. Cell targets were either homozygous wild-type orD32 CCR5 PBMCs. RT activity in the supernatant on day 5 was determined, and the results are shown as percent inhibition with respect to the control (con.) value. Error bars indicate standard deviations.

TABLE 3. HIV-2 infection of wild-type and

D

32/

D

32 PBMCs

a

Virus

RT activity (pg/ml) in supernatants after culture in PBMCs

Day 2 Day 5 Day 9 D32 wt D32 wt D32 wt

MIR

117

292

446

415

.

500

.

500

A-ND

264

283

441

328

.

500

.

500

ST

86

193

577

407

.

500

.

500

prCBL-20

72

121

309

477

.

500

.

500

CBL-20

112

108

484

.

500

.

500

.

500

prCBL-23

11

,

4

158

61

.

500

.

500

aVirus isolates were tested for their ability to infect PBMCs from an individual

who is homozygous for theD32 deletion in the CCR5 gene and from an indi-vidual homozygous for wild-type (wt) CCR5 gene. PBMCs were infected, and supernatants were analyzed for RT activity on days 2, 5, and 9.

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infection was restricted to particular coreceptors, cell-to-cell

fusion was induced by a broad range of chemokine receptors,

including CCR1, CCR2b, and CXCR2. The results of Bro¨n et

al. suggest that cell culture adaptation for CXCR4 use

prefer-entially restricts coreceptor use for virion-to-cell fusion

com-pared with cell-to-cell fusion and support the notion that the

physical requirements for these processes are subtly different

(6, 39).

So far, all primary and T-cell-line-adapted SI isolates of

HIV-1 use CXCR4 (2, 25, 54, 61). We tested the phenotypes of

the HIV-2 isolates described here by criteria used first for

HIV-1 (58) and later for HIV-2 (1). Of the primary isolates,

only MIR induced syncytia in PBMCs, while both MIR and

prCBL-23 induced syncytia in MT-2 cells. Of the four other

primary strains, ST and A-ND induced syncytia in at least one

of the T-cell lines tested, whereas V9 and prCBL-20 showed no

evidence of syncytium formation in any of the cell types tested.

Thus, we did not observe a clear correlation between CXCR4

use and syncytium induction in CXCR4

1

T cells by the HIV-2

strains used here.

The determination of coreceptor use by HIV-1 and HIV-2

strains in vitro, using CD4

1

cell lines, can be misleading (20).

Primary cell types that are targets for HIV infection in vivo

may express several coreceptors that can be used by HIV

strains in such in vitro assays. It is therefore hard to assess the

predominant coreceptor used on these cell types. It has been

shown previously that stimulated primary lymphocytes express

CCR1 (57), CCR2b (45), CCR3 (49), CCR4 (38), CCR5 (14),

CXCR4 (40), and BONZO (4, 36) as determined by mRNA

expression. In addition we have shown that CCR5

(unpub-lished data) and CXCR4 (40) are expressed at the cell surface

as detected by specific monoclonal antibodies. We assessed the

coreceptors used by HIV-2 strains A-ND, prCBL-20, and

CBL-20 on primary PHA- and IL-2-stimulated PBMCs by

test-ing whether the natural ligands for different coreceptors could

block infection. SDF-1 inhibited infection of PBMCs by all

three virus strains, while AOP-RANTES or MCP-1 had little

effect. Since SDF-1 has so far been shown to bind only to

CXCR4, these results suggest that this coreceptor is

predom-inantly used for PBMC infection.

We have shown that six primary strains of HIV-2 can use a

broad range of different coreceptors. Although the more

re-cently described coreceptors BOB, BONZO, and GPR1 were

not included in this study, it seems likely that they too will be

used by primary HIV-2 strains, at least for some

virus-core-ceptor combinations. How such a broad corevirus-core-ceptor use

influ-ences the cell types infected by HIV-2 strains in vivo remains

to be elucidated. Although HIV-2 is often described as less

pathogenic than HIV-1, many HIV-2-infected individuals do

progress to full-blown AIDS and suffer from encephalitis. It

will be of interest to establish whether the use of a wide

spec-trum of coreceptors available correlates with any disease state.

ACKNOWLEDGMENTS

We thank Robin Weiss and Graham Simmons for critical discussion

and reading of the manuscript. Dan Littman generously provided U87/

CD4 cells expressing different chemokine receptors. Erik de Clercq

and Dominic Schols kindly provided AMD3100. We also thank the

European Community Concerted Action Group on HIV Variability

for providing an intellectual forum for collaboration and discussion.

This work is funded by the Medical Research Council, United

King-dom, and supported partly by an EC Biomed II grant.

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TABLE 4. Tropism of primary HIV-2 isolates for cell lines

expressing human CD4

a

Virus

Infectivity (FFU/ml) with the following cell line:

HeLa/CD4 RD/CD4 SCL/CD4 WI-38T/CD4 U87/CD4/CXCR4

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aCells were seeded and infected as described in Materials and Methods. After

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bNT, not tested.

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on November 9, 2019 by guest

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Figure

TABLE 1. HIV-2 MIR infection of CCC/CD4 cells expressingdifferent chemokine receptors after transfection
TABLE 2. Phenotype determination of primary HIV-2 isolatesa
TABLE 3. HIV-2 infection of wild-type and �32/�32 PBMCsa
TABLE 4. Tropism of primary HIV-2 isolates for cell linesexpressing human CD4a

References

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