Primary Human Immunodeficiency Virus Type 2 (HIV-2) Isolates Infect CD4-Negative Cells via CCR5 and CXCR4: Comparison with HIV-1 and Simian Immunodeficiency Virus and Relevance to Cell Tropism In Vivo

Copyright © 1999, American Society for Microbiology. All Rights Reserved.

Primary Human Immunodeficiency Virus Type 2 (HIV-2) Isolates

Infect CD4-Negative Cells via CCR5 and CXCR4: Comparison

with HIV-1 and Simian Immunodeficiency Virus

and Relevance to Cell Tropism In Vivo

JACQUELINE D. REEVES,

* SAM HIBBITTS,

GRAHAM SIMMONS,

A

´ INE M

KNIGHT,

JOSE´ M. AZEVEDO-PEREIRA,

JOSE´ MONIZ-PEREIRA,

PAUL R. CLAPHAM

*

The Wohl Virion Centre, Department of Molecular Pathology, Windeyer Institute of Medical Sciences,

University College London, London, United Kingdom,

and Faculdade de

Farmacia, Universidade de Lisboa, Lisbon, Portugal

Received 1 April 1999/Accepted 7 June 1999

Cell surface receptors exploited by human immunodeficiency virus (HIV) and simian immunodeficiency

virus (SIV) for infection are major determinants of tropism. HIV-1 usually requires two receptors to infect

cells. Gp120 on HIV-1 virions binds CD4 on the cell surface, triggering conformational rearrangements that

create or expose a binding site for a seven-transmembrane (7TM) coreceptor. Although HIV-2 and SIV strains

also use CD4, several laboratory-adapted HIV-2 strains infect cells without CD4, via an interaction with the

coreceptor CXCR4. Moreover, the envelope glycoproteins of SIV of macaques (SIV

) can bind to and initiate

infection of CD4

cells via CCR5. Here, we show that most primary HIV-2 isolates can infect either CCR5

or CXCR4

cells without CD4. The efficiency of CD4-independent infection by HIV-2 was comparable to that

of SIV, but markedly higher than that of HIV-1. CD4-independent HIV-2 strains that could use both CCR5 and

CXCR4 to infect CD4

cells were only able to use one of these receptors in the absence of CD4. Our

observations therefore indicate (i) that HIV-2 and SIV envelope glycoproteins form a distinct conformation

that enables contact with a 7TM receptor without CD4, and (ii) the use of CD4 enables a wider range of 7TM

receptors to be exploited for infection and may assist adaptation or switching to new coreceptors in vivo.

Primary CD4

fetal astrocyte cultures expressed CXCR4 and supported replication by the T-cell-line-adapted

ROD/B strain. Productive infection by primary X4 strains was only triggered upon treatment of virus with

soluble CD4. Thus, many primary HIV-2 strains infect CCR5

or CXCR4

cell lines without CD4 in vitro.

CD4

cells that express these coreceptors in vivo, however, may still resist HIV-2 entry due to insufficient

coreceptor concentration on the cell surface to trigger fusion or their expression in a conformation

nonfunc-tional as a coreceptor. Our study, however, emphasizes that primary HIV-2 strains carry the potential to infect

CD4

cells expressing CCR5 or CXCR4 in vivo.

Human immunodeficiency virus type 2 (HIV-2) is endemic

in West Africa and has spread in the last decade to the west

coast of India (3, 43, 67), as well as causing numerous

infec-tions in Europe. The mortality rate following HIV-2 infection

is estimated to be a third lower than that for HIV-1 (84).

HIV-2 is closely related to simian immunodeficiency virus of

sooty mangabeys (SIV

) and SIV of macaques (SIV

).

SIV

is endemic and nonpathogenic in West African sooty

mangabey monkeys, even though high viral loads can

some-times be detected in plasma (65). The HIV-2 epidemic is likely

to have resulted from several zoonoses from wild SIV

-in-fected sooty mangabeys, and, consequently, primary HIV-2

strains are closely related by sequence to SIV

strains (30).

HIV and SIV are viruses with a lipid membrane that must

fuse with the cell membrane to allow the virus core and RNA

genome access to the cell cytoplasm. Glycoprotein spikes on

the surface of virus particles attach to specific receptors at the

cell surface and induce fusion of viral and cellular membranes.

HIV-1, HIV-2, and SIV strains interact with cell surface CD4

and seven-transmembrane (7TM) coreceptors to infect cells.

An interaction with CD4 triggers conformational changes in

gp120 allowing a secondary interaction with a 7TM molecule

to occur. The crystal structure of an HIV-1 gp120 core,

com-plexed with soluble CD4 (sCD4 [domains 1 and 2]) and a Fab

fragment of an antibody to a CD4-induced epitope, has been

solved (45). The 7TM receptor binding site is predicted to be

composed of conserved regions encompassing a bridging sheet

domain and residues within V3 (66, 88). CCR5 and CXCR4

are major coreceptors for HIV-1; however, there are marked

differences in coreceptor use between SIV and HIV-1. In

par-ticular, SIV

strains use CCR5 but not CXCR4, while other

coreceptors, including GPR15/BOB, STRL33/BONZO, and

GPR1, are more likely to be used (2, 15, 22, 28, 48, 49).

Previously we and others have shown that many primary and

laboratory-adapted HIV-2 strains can exploit a broad range of

coreceptors for infection of CD4

cell lines, including CCR5

and CXCR4 (9, 32, 51, 58, 78), while some primary HIV-2

strains from asymptomatic individuals predominantly use

CCR5 (32, 58, 78).

HIV-1 infection of CD4

cell cultures in vitro has been

extensively reported (for reviews, see references 12 and 13);

however, this is usually much less efficient than infection of

cells that express CD4. The relevance of CD4-independent

* Corresponding author. Mailing address: The Wohl Virion Centre,

Department of Molecular Pathology, Windeyer Institute of Medical

Sciences, University College London, 46 Cleveland St., London W1P

6DB, United Kingdom. Phone: 44 171-504 9562 or 44 171-504 9558.

Fax: 44 171-504 9555. E-mail: j.reeves@ucl.ac.uk or p.clapham@ucl.ac

.uk.

7795

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entry in vivo and its influence on pathogenesis are therefore

unclear. There is, however, evidence that CD4

brain

astro-cytes become infected by HIV-1 in vivo, particularly in

pedi-atric AIDS patients (68, 74). A CD4-independent variant of

HIV-1/IIIB selected by multiple passage in a CD4

T-cell line

was recently described. This virus utilized CXCR4 to infect

CD4

cells (36), yet substitution of the V3 loop with that from

the R5 BaL strain resulted in a virus capable of

CD4-indepen-dent infection via CCR5 (35). In contrast to HIV-1,

T-cell-line-adapted (TCLA) strains of HIV-2 can be readily T-cell-line-adapted to

infect a subset of CD4

human cell lines (14). This

CD4-independent infection occurs predominantly via CXCR4, most

likely reflecting the passage of these viruses through CXCR4

T-cell lines (27, 63). Low-level CD4-independent infection has

been reported for a single R5 HIV-2 isolate (11). For HIV-2

strains that are CD4 dependent, infection of CD4

cells is

often potently induced by prior treatment of virus particles

with sCD4 (14). Interestingly, recombinant envelope proteins

derived from some SIV

strains have been shown to

inter-act directly with CCR5 (26, 50), and infection of primary

CCR5

CD4

brain endothelial cultures has also been

re-ported (26).

Receptor use has profound implications for the cell tropism

and pathogenesis of HIV-2 strains in vivo. For instance, if

CD4-independent viruses occur or evolve in an infected

indi-vidual, then such strains are likely to be able to infect a broader

range of cell types at different sites in vivo. Moreover, the

conformation of the envelope glycoproteins that confer a

di-rect interaction with coreceptors may expose antigenic

epi-topes to neutralizing and other antibodies and thus influence

the capacity of the host to control viral replication. Here, we

show that many primary HIV-2 strains can infect CD4

cell

lines expressing either CCR5 or CXCR4. Primary cultures of

CD4

astrocytes were susceptible to infection by the TCLA

HIV-2 variant ROD/B. Intriguingly, however, primary X4

strains only infected astrocytes if virus was treated with sCD4.

These results indicate that CD4-independent infection of cell

lines observed in vitro may not reflect infection of CD4

cell

types in vivo or may require high levels of CXCR4 expression.

MATERIALS AND METHODS

Cells.Peripheral blood mononuclear cells (PBMCs), cultured in RPMI 1640 medium (GIBCO) supplemented with 20% fetal calf serum (FCS), 60␮g of penicillin and 100␮g of streptomycin per ml (pen/strep), were stimulated for 2 to 3 days with phytohemagglutinin (PHA; 0.5␮g/ml) and then cultured with interleukin-2 (IL-2; 20 U/ml) for 2 to 3 days prior to infection. T-cell lines H9 and Molt 4 were cultured in RPMI 1640 medium supplemented with 10% FCS and pen/strep. The human glioma cell lines U87, U87/CXCR4, U87/CD4, and U87/ CD4 cells stably expressing chemokine receptors CCR1, CCR2b, CCR3, CCR5, and CXCR4 (6, 22, 86), as well as NP2, NP2/CCR5, NP2/CD4, and NP2/CD4/ CCR5 (89) were cultured in Dulbecco’s modified Eagle’s medium (DMEM; GIBCO) supplemented with 5% FCS and pen/strep. The CD4⫺_human

rhabdo-myosarcoma cell line RD/TE671 (79); feline kidney cell lines CCC, CCC/ CXCR4, CCC/CD4, and CCC/CD4/CXCR4 (19, 77); and the human osteosar-coma cell line GHOST and the GHOST-derived lines expressing CCR1, CCR2b, CCR3, CCR5, and CXCR4 (10) were also cultured in DMEM supplemented with 5% FCS and pen/strep. Primary astrocytes prepared from fetal brain (83) were cultured in DMEM supplemented with 10% FCS, 20 mML-glutamine per ml, pen/strep, and 17.5␮g of neomycin per ml. Astrocyte cultures were positive for the astrocytic marker glial fibrillary acidic protein (GFAP), but negative for CD4 expression and the macrophage/microglial marker CD68. The use of fetal brain samples was approved by the Royal Marsden NHS Trust Research Ethics Committee and complied with institutional and ethical regulations. Fetal brains were obtained from the Medical Research Council Tissue Bank (Hammersmith Hospital, London, United Kingdom).

Viruses.Primary HIV strains were isolated from PHA- and IL-2-stimulated PBMCs derived from the peripheral blood of infected individuals. Isolates were minimally passaged in PBMCs from HIV-negative donors to prepare virus stocks. Stocks of TCLA viruses were produced from the CD4⫹_{T-cell lines H9 for}

HIV-1 and HIV-2 or Molt 4 for SIV strains. Table 1 lists all of the HIV and SIV strains used in this study and provides current information on the coreceptors

used by each strain to infect CD4⫹_{cell lines. The coreceptors used by primary}

HIV-2 strains ALI, MLC, TER, ETP, JAU, MIL, and SAB were characterized in this study.

Primary HIV-1 isolates.The CCR5 tropic (R5) primary HIV-1 isolates used include BR49 and BR92, from Brazilian patients (23), and SL-2 from a patient from Thailand (77), all of which were subtype B viruses isolated from asymp-tomatic individuals. The CXCR4 tropic (X4) viruses used include strains 2005 and 2044 (77); both subtype B viruses were isolated from patients registered in England with CD4 blood cell counts of⬍190 cells mm⫺3_{. R5/X4 viruses included} subtype B strains, 2028 and 2076, from English patients with CD4 counts of⬍190 cells mm⫺3_{(77). ACH-320.3.1.mc is a molecular clone of an isolate originating} from Amsterdam (31). HAN2 and HAN2-2mc (69) are a primary isolate and the

TABLE 1. HIV-1, HIV-2, and SIV strains

Virus strain

Coreceptor(s) used to infect CD4⫹_cells

(where known)b

R5/X4 Other(s)

HIV-2 Primary

MLC R5 (R1), (R3), (U87)

TER R5 R1, R3, U87

ALI R5, (X4) (R1)

ETP R5, X4 R1, R2b, R3

JAU R5, X4 R1, R2b, R3

MIR (R5), X4 (R1), (R2b), R3

prCBL-20 (R5), X4 R1, R2b, R3

prCBL-23 (R5), X4 (R1), R2b, R3

A-ND X4 R1, R2b, R3

MIL X4

SAB X4 (R3)

TCLA

CBL-23 R5, X4

ROD/A (pACR23) X4 R2b, R3, (CX3CR1)

ROD/B X4 R2b, R3, (CX3CR1)

CBL-20 X4

SIV

SIVAGMTYO-2 R5

SIVMAC251 R5

SIVMAC32H R5 R1, GPR15, STRL-33

SIVSMB670 R5

SIVSMg1010.2c R5 GPR15

SIVSMswg497c

HIV-1 Primary

BR49 R5

BR92 R5

SL-2 R5

2028 R5, X4 (R3), (R8), (STRL-33), (GPR-15)

2076 R5, X4 R3, R8, STRL-33, (GPR-15)

ACH320.3.1 R5, X4

HAN-2 R5, X4

HAN-2-2mc R5, X4

SL-12 R5, X4

2005 X4

2044 X4

TCLA

GUN-1 R5, X4

HXB2 X4

RF X4

a_{HIV and SIV strains used throughout this study are listed along with current} information on coreceptors used to infect CD4⫹_{cell lines (9, 23, 51, 63, 76, 77).} b_{Parentheses denote inefficient use of a coreceptor, where the infectivity titer} (FFU per milliliter) via this receptor is at least 2 logs lower than that of infection via either CXCR4 or CCR5 or less than 1 log above infection of parental cells. “U87” indicates that the parental U87/CD4 cells were susceptible without ex-pression of additional coreceptors.

c_{Reisolation of SIVSMB670 following infection of rhesus macaques.}

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corresponding molecular clone from Germany. SL-12 is a subtype E virus iso-lated from an asymptomatic individual from Thailand, at St. Mary’s Hospital, London, England.

TCLA HIV-1 viruses.The TCLA viruses included X4 HXB2 (62) and RF (60), as well as R5/X4 GUN-1 (80).

Primary HIV-2 isolates.Primary HIV-2 isolates described previously, includ-ing MIR (17), prCBL-20, prCBL23, and A-ND (51), use a broad range of coreceptors, including CCR1-3, CCR5, and CXCR4, to infect CD4⫹_{cells. MIR}

and prCBL-20 were isolated from Guinea-Bissau and Gambian AIDS patients respectively, while prCBL-23 and A-ND were from Gambian and Portuguese symptomatic individuals, respectively. Additional primary HIV-2 strains, char-acterized for receptor use in this study, were all isolated from Portuguese pa-tients with CD4 counts of⬍200 cells mm⫺3_{. ALI was isolated from a patient with} AIDS-related complex. TER, JAU, MIL, and SAB were from AIDS patients, and MLC and ETP were from symptomatic patients.

TCLA HIV-2 viruses.The TCLA X4 HIV-2 strains included ROD/A, which was generated from the CD4-dependent, infectious proviral clone of ROD, pACR23 (38). ROD plasmid DNA was transfected into RD cells, and virus progeny were seeded onto H9 cells to produce virus stocks (64). ROD was the first reported isolate of HIV-2 which originated from the Cape Verde Islands, Senegal (16). ROD/B is a CD4-independent variant derived from ROD/A fol-lowing passage through C8166 cells (14). Other TCLA HIV-2 strains included CBL-20 and CBL-23 (73), which are derived from the primary isolates prCBL-20 and prCBL-23, respectively (described above). Stocks of CBL-20 and CBL-23 were produced from H9 cells.

SIV strains. The R5 TCLA SIV strains used were SIVMAC251 (21), SIVMAC32H (18), SIVSMB670 (56), and SIVAGMTYO-2 (4). G1010.2 and swg497 are reisolations of SIVSMB670 following infection of rhesus macaques (kindly provided by M. Murphey Corb).

Infectivity assays.Cells were seeded into 48-well trays on the day prior to infection, at 1⫻104_{cells/well for U87, NP2 cells, and derivatives and 4⫻10}4 cells/well for astrocytes, CCC cells, and derivatives. Infections were performed in duplicate, or with serial dilutions of 100␮l of cell-free virus supernatant in the absence or presence of 5␮g of baculovirus-derived sCD4 per ml. Virus was incubated with cell lines for 3 h before addition of 500␮l of growth medium. Cells were immunostained for virus expression 4 days postinfection. Astrocytes were challenged with 5 ⫻103_{focus-forming units (FFU) of each virus (as} measured on U87/CD4/CCR5 or U87/CD4/CXCR4 cells). Viral supernatant was removed from infected astrocytes 16 h postinfection, and cells were washed four times before addition of 500␮l of culture medium. Supernatants, sampled over 26 days, were assayed for reverse transcriptase (RT) activity by an enzyme-linked immunosorbent assay (Retrosys RT activity kit; Cavidi Tech, Uppsala, Sweden). Following the final harvest of supernatant for RT analysis, astrocytes were immunostained for viral antigen expression.

Receptor ligands, tested for their ability to inhibit HIV-2 ROD/B infection of primary astrocytes, included CXCR4 ligand SDF-1␣(7, 57) and the CXCR4-specific monoclonal antibody (MAb) 12G5 (27, 53) and CCR5 ligand RANTES (70) and the CD4-specific MAb Q4120, which binds domain 1 and interferes with gp120 binding (33). Briefly, primary fetal astrocytes were preincubated with ligands at 2⫻final concentration for 1 h before an equal volume of virus was added for 3 h. Cells were then washed three times in growth medium, and 500␮l of medium was replaced. Cultures were incubated for 3 days, fixed in methanol-acetone (1:1), and immunostained for viral antigens.

Immunostaining.HIV-1-infected cells were immunostained for p24 antigen as previously described (14). HIV-2-infected cells were fixed for 10 min in metha-nol-acetone (1:1). Cells were then immunostained with serum pooled from six HIV-2⫹ _{individuals (World Health Organization panel C) at a dilution of}

1:4,000. SIV-infected cells were immunostained with HIV-2 serum (as described above) or with a mixture of SIV envelope MAbs, KK7a and KK41 (39, 40). ␤-Galactosidase conjugates of antihuman or antimouse antibodies (Southern Biotechnology Associates, Inc. [dilution 1:400]) were used to detect first-layer antibodies, as appropriate. Infected cells were immunostained blue with addition of 5-bromo-4-chloro-3-indolyl-␤-galactopyranoside (X-Gal; 0.5 mg/ml in phos-phate-buffered saline [PBS] containing 3 mM potassium ferricyanide, 3 mM potassium ferrocyanide, and 1 mM magnesium chloride) as previously described (14). Individual or groups of blue-stained cells were regarded as foci of infection, and virus infectivity was estimated as FFU per milliliter.

Comparison of CD4-independent infection by HIV-1, HIV-2, and SIV. Infec-tivity titers (FFU per milliliter) for 14 HIV-1, 15 HIV-2, and 6 SIV strains were determined on CD4⫺_{cells in the presence and absence of sCD4 and compared}

to infectivity titers on CD4⫹_{cells. Ratios of infectivity for CD4}⫺_{and CD4}⫹_cells

were calculated from virus titrations of CCC and CCC/CD4 cells transfected with either CCR5 or CXCR4 expression vectors. Ratios for some strains were deter-mined from titers on the stable cell lines NP2/CCR5 and NP2/CD4/CCR5 and on CCC/CXCR4 and CCC/CD4/CXCR4. Background infectivity on corresponding coreceptor-negative cells was subtracted. A ratio of 1 indicates equivalent infec-tion on CD4⫺_{and CD4}⫹_{cells, while a ratio of 0.1 implies a 10-fold-less-efficient}

infection of CD4⫺_{compared to that of CD4}⫹_cells.

Determination of cell surface receptor expression.Primary astrocyte cultures were analyzed for cell surface expression of CD4, CXCR4, and CCR5 by flow cytometry. CXCR4 expression on astrocytes was compared to that on CCC/ CXCR4 and U87/CXCR4 cells. Cells (3⫻105_{; preincubated in PBS–1% FCS–}

0.05% sodium azide for 30 min) were incubated with MAb 12G5 (27, 53) (2 ␮g/ml) to detect CXCR4 expression, MAb 2D7 (2␮g/ml) (87) to detect CCR5 expression, or an anti-CD4 domain 4 R-phycoerythrin conjugate (CD4 D4 R-PE; 1:10 dilution [Becton Dickinson]) to detect CD4 expression, as well as the appropriate isotype controls diluted in 100␮l of PBS–1% FCS–0.05% sodium azide for 1 h at room temperature. Cells incubated with 12G5, 2D7, and isotypes were washed twice in PBS–1% FCS–0.05% sodium azide before resuspension in 100␮l of antimouse immunoglobulin G (IgG) conjugated with fluorescein iso-thiocyanate (FITC; 1:40 dilution [DAKO]) for 30 min. All cells were then washed once in PBS–1% FCS–0.05% sodium azide, twice in PBS–0.05% sodium azide, resuspended in 100␮l of PBS–0.05% sodium azide, and added to 300␮l of formal saline (4% of formaldehyde in 0.5% NaCl and 1.5% Na2SO4) before analysis by flow cytometry.

Immunostaining for envelope expression in ROD/B-infected astrocytes. As-trocytes were seeded, the day prior to infection, onto 8-well chamber slides (Nunc) at 4⫻104_{cells/well. Cells were infected with 100␮l of virus supernatant} for 4 h before addition of 500␮l of culture media. Seven days postinfection, cells were fixed in methanol-acetone (1:1; 5 min), washed in PBS–1% FCS, and incubated with antibodies to HIV-2 envelope and GFAP (rat MAb 44.2g [52]; rabbit anti-cow GFAP [DAKO], 1:100). Bound primary antibodies were detected with anti-rat IgG R-PE (1:120 [Harlan Sera-Labs]) or swine F(ab⬘)2anti-rabbit FITC (1:15 [DAKO]). Immunostained cells were visualized with a fluorescent microscope.

RESULTS

HIV-2 coreceptor use on CD4

cells.

Table 1 lists the HIV-1,

HIV-2, and SIV strains used in this study and known

corecep-tors used for infection of CD4

cell lines. We showed

previ-ously that several primary HIV-2 isolates use a broad range of

coreceptors, including CCR5 and CXCR4 (51). A further

sev-en primary HIV-2 isolates were analyzed for coreceptor use.

Two of these (ETP and JAU) could utilize a range of

recep-tors, including CCR1, CCR2b, CCR3, CCR5, and CXCR4. A

further isolate (TER) could infect CD4

cells expressing CCR1,

CCR3, and CCR5. Two strains were identified that

predomi-nantly use CCR5 (ALI and MLC), and two were identified that

predominantly or exclusively use CXCR4 (MIL and SAB).

Coreceptor use was assessed by testing infection of a set of

U87/CD4 cell lines that individually express CCR1, CCR2b,

CCR3, CCR5, and CXCR4 (6, 22). Where possible, results

were confirmed by using the panel of GHOST/CD4/coreceptor

cell lines (10) or CCC/CD4 cells transfected with and

tran-siently expressing each coreceptor. Assessment of coreceptor

use by some HIV-2 strains is complicated by use of

unidenti-fied coreceptors expressed naturally on U87/CD4 and CCC/

CD4 cells, while GHOST/CD4 cells express low levels of

CXCR4 that result in background infectivity. For instance,

TER infects the parental U87/CD4 cell line without expression

of exogenous coreceptors; however, infection was increased

30- to 100-fold on U87/CD4 cells expressing CCR5, CCR3, and

CCR1 (Table 1).

Primary HIV-2 infection of CD4

cells expressing either

CCR5 or CXCR4.

We tested CD4-independent infection for

seven primary HIV-2 isolates, including two X4 strains (SAB

and MIL), two R5 strains (ALI and MLC), and TER, ETP, and

JAU, which use a broad range of coreceptors for infection of

CD4

cells (Table 1). Infection was compared to that of the

R5 SIV

32H strain and two X4 TCLA strains of the HIV-2

ROD isolate: ROD/A, which is mainly CD4 dependent; and

ROD/B, which efficiently infects CXCR4

CD4

cell lines

(63).

The CD4

cell lines currently available that stably express

high levels of either recombinant CXCR4 or CCR5 are CCC/

CXCR4 (feline kidney), U87/CXCR4, and NP2/CCR5 (human

gliomas). Figure 1A shows infectivity for CCC/CXCR4

com-pared to infection of the counterpart CCC/CD4/CXCR4 cell

line, while Fig. 1B shows infectivity titers for NP2/CCR5 and

NP2/CD4/CCR5. We also assessed the effect of sCD4 on

in-fectivity for CD4

CCC/CXCR4 and NP2/CCR5 cells.

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stantial CD4-independent infection was recorded for ETP,

MIL, and SAB on CCC/CXCR4 cells (Fig. 1A). Infection by

these three strains was enhanced between 10- and 100-fold by

sCD4. As expected, ROD/B infected CD4

CCC/CXCR4 cells

as efficiently as the equivalent CD4

cells. ROD/A infectivity

was about 1,000-fold less efficient without CD4 (accounting for

background infection), but was enhanced 100-fold following

sCD4 treatment. Other strains either infected only CD4

cells

or failed to use CXCR4 as a coreceptor and were unaffected by

sCD4. As expected, no infection of the CCC/CXCR4 cells by

R5 strains was detected. Similar results were obtained

follow-ing infection of CD4

U87/CXCR4 cells (data not shown).

Primary HIV-2 strains that use CCR5 on CD4

cells ranged

from ALI (predominantly CCR5) to strains that used multiple

coreceptors (Table 1). On CD4

NP2/CCR5 cells, infection by

three strains, ALI, JAU, and TER, was observed (Fig. 1B).

Infection was comparable with that for SIV

32H. sCD4

enhanced infection of NP2/CCR5 by these four strains as well

as inducing infection by MLC.

Inhibition experiments with receptor ligands (AMD3100,

specific for CXCR4 [24, 71, 72]); AOP-RANTES, a potent

inhibitor of infection via CCR5 (75); and Q4120 and 5A8,

MAbs specific for CD4 (33, 55) confirmed that CXCR4 and

CCR5 were used for CD4-independent infection, but not CD4

(data not shown).

Comparison of CD4-independent infection by HIV-1, HIV-2,

and SIV.

Infectivity titers of 14 HIV-1, 15 HIV-2, and 6 SIV

strains were compared for CD4

and CD4

cells expressing

either CCR5 or CXCR4. These strains included R5 and X4

viruses as well as viruses that used a range of different

core-ceptors, including both CCR5 and CXCR4 (Table 1). Figure 2

shows infectivity ratios for each strain calculated as the

infec-tivity titer (FFU per milliliter) for CD4

cells divided by the

infectivity titer for the equivalent CD4

cells. Although several

viral strains could use both CCR5 and CXCR4 to infect CD4

cells, CD4-independent infection occurred only via one of

these receptors. Ratios for either CCR5

or CXCR4

cells are

therefore shown (detailed as R5 or X4), as is infection with or

without sCD4 treatment. Ratios for HIV-2 strains were of the

same order as those for SIV strains, while HIV-1 ratios were

substantially lower, with few strains able to infect CD4

CCC

cells that expressed CXCR4 and none able to infect CD4

CCR5

CCC cells. Only one primary HIV-1 strain, 2005, could

infect CXCR4

CCC cells, and infection was enhanced by

sCD4. Another primary strain (HAN-2) and the TCLA strains

RF and GUN-1 also infected CXCR4

CCC cells, but only

if treated with sCD4. Interestingly, RF infected CD4

U87/

CXCR4 cells without sCD4 (not shown), highlighting the cell

type specificity of receptor dependence for this virus. These

results show that primary HIV-2 strains, like SIVs, are

sub-stantially less reliant on CD4 for infection than HIV-1

strains.

Seven HIV-2 strains (JAU, MIR, ETP, ALI, prCBL-23,

CBL-23, and prCBL-20) used both CCR5 and CXCR4 to

in-fect CD4

cells (Table 1). In the absence of CD4, however,

ETP and prCBL-23 could only use CXCR4 and JAU and ALI

could only use CCR5, while MIR, prCBL-20, and CBL-23 used

neither CCR5 nor CXCR4 efficiently. CD4-independent

infec-tion was mediated by the receptor preferentially used on CD4

cells.

Susceptibility of CXCR4

primary fetal astrocyte cultures

to CD4-independent infection by HIV-2.

Primary cultures of

CD4

fetal astrocytes were analyzed for CXCR4 and CCR5

expression. Figure 3 shows flow cytometric analysis of

astro-cytes immunostained with 12G5, a CXCR4-specific MAb, and

indicates astrocytes expressed CXCR4, but at lower

concen-trations compared to U87/CXCR4 and CCC/CXCR4 cells.

Astrocytes were negative for CD4 and CCR5 expression, as

assessed by Q4120 and 2D7 immunostaining (data not shown).

The susceptibility of astrocytes to CD4-independent

infec-tion was determined. Figure 4 shows syncytia in an astrocyte

culture infected with ROD/B. Costaining for the astrocyte

marker, GFAP, and for gp120 indicated that late gene

expres-sion is evident in ROD/B-infected astrocytes. ROD/B infection

of astrocytes was inhibited by the CXCR4 ligands MAb 12G5

and SDF-1, but not by the CD4-specific MAb Q4120 or the

CCR5 ligand RANTES (Fig. 5). These results confirm that

CD4-independent infection was mediated via CXCR4.

Astro-cytes, purified from two independent fetal brain samples, were

then challenged with a panel of HIV strains by using equivalent

infectivity doses for each, in the presence and absence of sCD4

(Fig. 6). Infectivity doses for astrocytes were assessed as FFU

on U87/CD4/CXCR4 or U87/CD4/CCR5 cells. The HIV-2

strains tested included the two primary HIV-2 X4 strains, SAB

and MIL; the R5 strain, TER (which uses CCR5, CCR1, CCR3

and other coreceptors); and the TCLA viruses ROD/A and

ROD/B. We also included the primary HIV-1 X4 strain, 2005,

which infected CD4

CCC/CXCR4 cells, albeit inefficiently.

Replication and virus production were assessed by testing

su-pernatants for RT activity over 26 days, after which cells were

fixed and immunostained for viral antigens by using HIV-2

FIG. 1. Infection of CD4⫺_{and CD4}⫹_{cells stably expressing either CCR5 or}

CXCR4 by primary isolates of HIV-2. Infectivity titers of primary HIV-2 isolates on CD4⫺_{cells (⫹sCD4 or⫺sCD4) and CD4}⫹_{cells are compared to those of}

TCLA HIV-2 (ROD/A and ROD/B) and SIV (MAC32H). Results are repre-sentative of at least three experiments with titers expressed as FFU per milli-liter⫹standard deviation. (A) Infection of CCC/CXCR4 and CCC/CD4/CXCR4 cells, showing substantial CD4-independent infection by MIL, SAB, ETP, and TCLA ROD/B on CXCR4⫹_{cells. (B) Infection of NP2/CCR5 and NP2/CD4/}

CCR5 cells showing CCR5-mediated CD4-independent infection by JAU, TER, and ALI as well as SIVMAC32H. Asterisks denote background infection in which virus infects CD4⫹_{parental cells in the absence of additional coreceptor expression.}

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human serum. Figure 6 shows that ROD/B productively

in-fected astrocytes from two fetal brains, albeit rather modestly.

The two primary X4 strains, MIL and SAB, only showed

pos-itive replication if first treated with sCD4, indicating that

as-trocytes support postentry replication by primary HIV-2

strains. Astrocyte culture 2 expressed slightly higher levels of

CXCR4 (Fig. 3) and when infected produced higher levels of

supernatant RT compared to astrocyte culture 1 (Fig. 6B and

A, respectively). These results suggests that susceptibility to

infection may correlate with CXCR4 concentration on the cell

surface. ROD/A replication was detected if astrocytes were

challenged with a 20-fold-higher dose of virus infectivity, but

only following sCD4 treatment, while 2005 required 1,000-fold

more virus to initiate productive infection (data not shown).

No replication was observed for the R5 HIV-2 isolate TER.

Immunostaining of fixed astrocytes for viral antigens after 26

days of culture showed that the presence of infected cells

correlated with detection of RT activity in the cell supernatant.

Interestingly, although similar levels of RT activity were

de-tected for ROD/B (without sCD4) as for MIL and SAB (with

sCD4), many more ROD/B-infected astrocytes were observed

by immunostaining at day 26 postinfection (Fig. 6C). The fewer

SAB- or MIL-infected cells must produce more progeny

viri-ons than cells infected by ROD/B.

We determined if virus released from astrocytes was

infec-tious by plating supernatants on U87/CD4 cells expressing

either CXCR4 or CCR5. MIL and SAB, rescued from

sCD4-induced astrocyte infections, and ROD/B, rescued from

astro-cytes infected in the absence or presence of sCD4, were fully

infectious for U87/CD4/CXCR4 cells. No infectious virus was

rescued from TER-infected astrocytes or from astrocytes

in-FIG. 3. Surface expression of CXCR4 on primary fetal astrocytes and CCC/ CXCR4 and U87/CXCR4 cells. Flow cytometric analysis using 12G5 (CXCR4-specific MAb) and isotype (IgG2a MAb) shows a low level of cell surface CXCR4 on astrocytes from two fetal brains (top), compared to those of CCC/CXCR4 and U87/CXCR4 cells (bottom).

FIG. 2. Comparison of CD4-independent and CD4-dependent infection by HIV-1, HIV-2, and SIV. Infectivity plotted as ratios between titers on CD4⫺_cells

(⫺sCD4 or⫹sCD4) and CD4⫹_{cells expressing either CCR5 or CXCR4 (as described in Materials and Methods). The coreceptor used by R5X4 viruses for}

CD4-independent infection is denoted in parentheses after the strain designation. The results represent at least two experiments for each virus.

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fected with 2005 or ROD/A at doses equivalent to that of

ROD/B (data not shown).

DISCUSSION

Some T-cell-line-passaged HIV-2 strains infect CXCR4

cells without CD4 (27, 63). The first CD4-independent variant

we identified (ROD/B) emerged spontaneously from a T-cell

line chronically infected with the prototype HIV-2

strain

(14). The ROD/B envelope retains the capacity to interact with

CD4, but can efficiently utilize CXCR4 alone for infection of

CD4

cells (63). Only two amino acid substitutions in the

envelope (one at the base of the V4 loop and one near the

leucine zipper-like domain in the transmembrane) were

re-quired to confer CD4 independence on ROD, although further

changes (in the V3 loop and at the base of V4) increased the

efficiency of infection without CD4 (64). Whether ROD/B-like

strains evolve or exist in vivo and whether CD4-independent

infection influences HIV-2 tropism or pathogenesis has been

unclear. We show here, however, that many primary HIV-2

isolates can infect CD4

cells via human CCR5 or CXCR4.

CD4-independent infection via CCR5 was at levels similar to

those of the CD4-independent SIV strains. Rhesus CCR5 has

been shown to function more efficiently than human CCR5 as

a primary receptor for SIV (25), but was not utilized in these

studies. No HIV-1 strains were found to use CCR5 in the

absence of CD4 (Fig. 2), although one primary HIV-1 X4

isolate (2005) infected CD4

CCC cells via CXCR4. To assess

whether CD4-independent and sCD4-induced infection of

CD4

cell lines was relevant for in vivo replication, we tested

if primary HIV-2 isolates infected primary CXCR4

fetal

as-trocyte cultures. Only the TCLA HIV-2 strain, ROD/B,

in-fected primary astrocytes. The primary X4 strains, MIL and

SAB, both of which efficiently infect CXCR4

CCC and U87

cells without CD4, did not infect the astrocyte cultures,

al-though infection was induced by sCD4. These results

demon-strate that the capacity of HIV-2 strains to infect CD4

cells is

profoundly influenced by cell type and determined by the

con-centration/presentation of cell surface coreceptors and/or by

currently unidentified cell surface factors.

For HIV-1, non-syncytium-inducing/R5 strains are usually

transmitted. Syncytium-inducing (SI) strains that use CXCR4

can be isolated from about 50% of AIDS patients, and their

emergence correlates with a more rapid decline in numbers of

CD4

T-cells (42). Such SI viruses either can use a range of

coreceptors, including CCR5 and CXCR4, or alternatively

seem to be specific for CXCR4 (76). Similarly, HIV-2 isolates

that use mainly CCR5 and not CXCR4 have been identified

(32, 58, 78); however, the majority of isolates use a broad range

of coreceptors, including CCR5 and CXCR4 as well as

core-ceptors rarely used by HIV-1 (e.g., CCR1) (9, 32, 51, 58, 78).

Two primary HIV-2 isolates studied here used CXCR4 only or

predominantly, yet few such HIV-2 strains have been reported

previously (32). These two X4 viruses were proficient for

in-fection of CD4

CXCR4

cell lines.

HIV-1 strains that use both CCR5 and CXCR4 (R5X4)

interact differently with CCR5 compared to R5 viruses.

CCR5-dependent infection by R5X4 strains is especially sensitive

both to CCR5 amino acid substitutions (5, 59) and to inhibition

by the

␤

-chemokine RANTES (41). Thus, evolution of HIV-1

from R5 to R5X4 seems to compromise the interaction of the

viral envelope with CCR5. Here, CD4-independent infection

by HIV-2 R5X4 strains indicated a spectrum of phenotypes,

none of which were able to use both CCR5 and CXCR4. Of

seven R5X4 strains, one used CCR5 only and three used

CXCR4 only, while the three others used neither CCR5 nor

FIG. 5. CD4-independent infection of astrocytes is inhibited by CXCR4 li-gands. (A) Inhibition of ROD/B infection by the CXCR4 ligand SDF, but not the CCR5 ligand RANTES. (B) Inhibition of ROD/B infection by the CXCR4-specific MAb 12G5, but not the CD4-CXCR4-specific MAb Q4120. These results are consistent with CD4-independent infection of astrocytes via CXCR4. Results are representative of at least three experiments, with titers expressed as FFU per milliliter.

FIG. 4. Envelope expression and syncytium formation in astrocytes infected by ROD/B. Immunofluorescence of HIV-2 ROD/B-infected fetal astrocyte cultures. (A) Expression of the astrocyte marker GFAP (green). (B) Expression of ROD/B envelope in astrocyte syncytia (red). (C) Overlay of GFAP and envelope antigen staining.

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CXCR4 efficiently for CD4-independent infection. It is

possi-ble therefore that CD4-independent infection by these R5X4

strains reflects an evolution from high-CCR5–low-CXCR4,

low-CCR5–low-CXCR4 to low-CCR5–high-CXCR4 affinity.

For these strains, interaction with CD4 presumably overrides

lower env-7TM interactions and increases the range of

core-ceptors available for infection.

It is uncertain why HIV usually needs two coreceptors to

enter cells, nor is it clear whether other lentiviruses or

retro-viruses use one or two receptors. Single receptors have been

identified for murine leukemia virus (MLV); the

14-transmem-brane cation transporter for ecotropic MLV (1) and the

10-transmembrane phosphate transporter, Pit-2, for amphotropic

MLV (54). Gibbon ape leukemia virus and feline leukemia

virus both use the related phosphate transporter, Pit-1 (37, 81).

Avian leukosis subgroup A (ALV-A) viruses use a receptor

related to the low-density lipoprotein receptor (90), while

sub-groups B and D share a tumor necrosis factor receptor-like

molecule (8). Although it seems likely that these receptors are

sufficient to trigger virus entry and replication, only for ALV-A

is there direct evidence that the identified receptor alone is

needed (20, 34). Willett et al. (85, 86) showed that

cell-line-adapted strains of feline immunodeficiency virus use CXCR4

(either feline or human CXCR4) for entry, but so far no other

receptor equivalent to CD4 has been identified.

We speculate that the viral ancestors of HIV and SIV

orig-inally used a 7TM receptor alone. Acquisition of a second

receptor such as CD4 may have provided selective advantages

to a virus that persistently replicates in the face of a vigorous

host immune response. Variation in the envelope must help

the virus to escape from neutralizing antibodies, but too much

divergence will inevitably weaken the envelope-7TM

interac-tion and reduce the efficiency of infecinterac-tion. On the HIV-1

envelope, the gp120 site for binding the 7TM receptor is

X4 HIV-2 strains MIL and SAB, primary R5 HIV-2 strain TER, TCLA X4 HIV-2 strains ROD/A and ROD/B, and primary X4 HIV-1 strain 2005. Infections were performed in the presence and absence of sCD4. Replication was assessed by RT activity in supernatants determined on duplicate wells. (C) Astrocytes infected with ROD/B, MIL, and SAB (⫺sCD4 or⫹sCD4) and immunostained for HIV-2 antigens.

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posed only after CD4 is contacted. This mechanism may

en-able potential neutralizing epitopes on or around the 7TM

binding site to be hidden until the fusion reaction is triggered,

and perhaps even then. Our results suggest that for HIV-2 and

SIV, the envelope glycoproteins form a subtly different

con-formation compared to HIV-1, where the 7TM binding site on

gp120 is at least partially exposed or formed, enabling direct

contact without CD4. The role of CD4 binding for these strains

is currently unclear but may (i) modify the 7TM binding site to

increase the affinity of the env-7TM interaction, or (ii)

con-tribute extra energy or a “kick” to the env-7TM contact needed

to trigger fusion of viral and cell membranes. Either or both of

these roles would provide HIV-2 with the capacity to exploit

coreceptors that otherwise do not interact with gp120 strongly

enough to trigger fusion.

Astrocytes do not express CD4 yet become infected in vivo,

at least in pediatric HIV-1 AIDS cases (68, 74). Such infection

is relatively unproductive, with structural

gag

and

env

genes

poorly expressed. Coreceptors used for infection of astrocytes

have not been identified, although glial cell lines, e.g., U87,

NP2 and U373, do not usually express CXCR4 or CCR5. The

primary fetal astrocytes used in this study, however, were

pos-itive for CXCR4 and supported replication by ROD/B, thus

demonstrating the potential of such cells to support replication

in vivo. The lack of astrocyte infection by primary X4 HIV-2

strains, in the absence of sCD4, may be due to the relatively

low level of CXCR4 expression on astrocytes compared to that

CXCR4

CCC and U87 cell lines, although Edinger et al.

recently reported that CD4-independent infection by SIV

strains required only a low level of CCR5 (25). Alternatively,

CXCR4 may be present on astrocytes in a different

conforma-tion than that found on the cell lines examined in this paper, as

recently shown for CCR5 on different cell types (47). It has

also been shown that CXCR4 may exist mainly as oligomers in

macrophages, compared to monomers in monocytes, which

may influence coreceptor activity (46). Additionally,

posttrans-lational modifications such as glycosylation or sulfation may

affect the efficiency of coreceptor utilization (29, 61, 82). Our

results showing CD4-independent infection by primary HIV-2

strains on cell lines in vitro should therefore be interpreted

with care until further studies are done to elucidate the cell

types that are infected by HIV-2 in vivo. We cannot rule out a

very low level of infection of astrocytes by primary HIV-2

isolates, because PCR detection or coculture with susceptible

cell types was not attempted. Whether mechanisms analogous

to sCD4-induced infection occur in vivo is unknown, although

soluble forms of CD4 have been detected in serum (44).

As-trocytes represent only one cell type that is a potential target

for HIV-2 infection in vivo. Other CD4

cell types expressing

either CCR5 or CXCR4 may behave more like the CCC, U87,

or NP2 cell lines shown here to be susceptible to HIV-2

infec-tion without CD4. Our observainfec-tions, however, show clearly

that primary HIV-2 isolates (as for SIV strains) carry the

potential to infect CD4

cells in vivo via an interaction with

CCR5 or CXCR4 that bypasses CD4.

ACKNOWLEDGMENTS

We thank Robin Weiss for continuing encouragement and for

crit-ical reading of the manuscript, M. H. Lourenc¸o for HIV-2 isolates ETP

and MLC, and K. Mansinho for patient information. We also thank

Hiroo Hoshino and Yasushi Soda, (University of Gunma, Japan) for

kindly providing NP2 cells, Dan Littman for GHOST and U87 cells,

Michael Murphey Corb for SIV

viruses, Jim Hoxie (University of

Pennsylvania), for MAb 12G5, Amanda Proudfoot (Serono

Pharma-ceutical Research Institute, Switzerland) for chemokines, and the

Medical Research Council Tissue Bank, Hammersmith Hospital, for

fetal brain samples. We are grateful to Hilton Whittle, Koya Ariyoshi,

and Tom Blanchard (MRC Laboratories, The Gambia), as well as

Yasu Takeuchi and Massimo Pizzato for helpful discussions; and we

thank Garry Francis and Harvey Holmes at the MRC AIDS reagent

project for providing many of the reagents used in this study.

Our HIV research is funded by the Medical Research Council,

United Kingdom, and partly by an EC Biomed II grant. Research

performed in the laboratory of J.M.-P. was supported by Comissa˜o

Nacional da Luta a SIDA and contract PRAXIS N/2/2.1/SAU/16/94.

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