INSERM Unité 380 Laboratoire de Génétique et Pathologie Expérimentales, Institut Cochin de Génétique Moléculaire, 22 rue Méchain, 75014 Paris, France1
Université Paris 7 Denis Diderot, UFR de Biochimie, 75251 Paris Cedex 05, France2
Author for correspondence: Uriel Hazan (at INSERM).Fax +33 1 40 51 64 07. e-mail hazan{at}icgm.cochin.inserm.fr
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Abstract |
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Introduction |
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A few HIV isolates can use simian (rhesus and fascicularis monkey) (Himathongkham & Luciw, 1996 ), murine (Bieniasz et al., 1997
; Tachibana et al., 1997
), feline (Willett et al., 1997
) or rat (Pleskoff et al., 1997a
) CXCR4 when the receptor is expressed in transfected human CD4-positive, CXCR4-negative cells. No HIV enters the simian cell line COS-7, derived from African green monkey (agm), under normal conditions of co-receptor expression. The nature of the co-receptor, the cell type producing the co-receptor and the presence of human (h-) CD4 seem to be the most important factors determining virus entry (Dimitrov, 1997
).
While CD4 is almost always required, two CD4-independent HIV isolates have been characterized after long-term culture, the HIV-2 Rod/B isolate (Clapham et al., 1992 ; Reeves & Schulz, 1997
) and the HIV-1 m7NDK virus (Dumonceaux et al., 1998
). This CD4-independent entry is due to mutations in the env gene, and both mutants use CXCR4 as receptor (Dumonceaux et al., 1998
; Endres et al., 1996
; Reeves et al., 1997
).
We have shown previously that a cluster of mutations in the C2, V3 and C3 regions of the m7NDK env gene are responsible for its ability to enter CD4-negative cells. We have proposed that these mutations in gp120 increase the affinity of the virus for CXCR4, thus allowing CD4-independent membrane fusion. This present paper describes the characterization of the m5NDK virus, which can fuse with simian COS-7 cells expressing h-CD4. This virus contains five of the seven mutations in m7NDK and these five mutations are responsible for its phenotype. The two other mutations in C2 in the env gene of m5NDK (which leads to m7NDK) result in a virus that can fuse with CD4-negative COS-7 cells. Expression of agm-CXCR4 by the non-permissive human U373MG cell line demonstrated that this protein is used as a co-receptor by m5 and m7NDK viruses. Fusion blockage experiments with the CXCR4-specific ligand SDF-1 supported this conclusion. We have also shown that the wild-type (wt) NDK virus, which cannot normally fuse with h-CD4-positive COS-7 cells, can do so only when agm-CXCR4 is overproduced in these cells. These results indicate, therefore, that the amount of expressed co-receptor is a major determinant of virus entry and that the five mutations in the env gene of m5NDK are sufficient to circumvent the very low virus fusion efficiency that results at the normal level of expression of the co-receptor.
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Methods |
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m5NDK and m7NDK viruses and the chronically infected CEM or HeLa cell lines have been described previously (Dumonceaux et al., 1998 ).
Plasmid constructs.
Plasmid pLTRLuc has been described previously (Dumonceaux et al., 1998 ). The LTRLuc-PGK-hygro plasmid contains a luciferase gene under the control of the SacIHindIII long terminal repeat (LTR) fragment of HIV-1 NDK isolate and the hygromycin B resistance gene under the control of the phosphoglycerate kinase promoter (Adra et al., 1987
), both cloned into pBluescript SK(+) (Stratagene). The h-CXCR4 expression plasmid was a gift from B. Moser (University of Bern, Switzerland) (Loetscher et al., 1994
). The agm-CXCR4 sequence was amplified by RTPCR and cloned into the pcDNA3 plasmid (Invitrogen) using the HindIII and XbaI sites. The pCMV-CD4 plasmid was constructed by inserting the CD4 coding sequence downstream of the CMV promoter using the EcoRI and BamHI sites of the pUHD15-1 plasmid (Gossen & Bujard, 1992
) and an EcoRIPvuI-digested SV40puromycin resistance gene cassette was subcloned into the PvuI and HindIII sites of pCMV-CD4 expression plasmid. pPGK-hygroR was a gift from M. Alizon (ICGM, Paris). Replacing the hygromycin resistance gene with the CD4 cDNA resulted in pPGK-CD4.
RTPCR.
agm-CXCR4 was obtained after RTPCR performed on COS-7 cell RNA. Total RNA was extracted from 5x106 COS-7 cells by using a commercial kit (Bioprobe, RNAB). Reverse transcription was performed on 10 µg total RNA by using the AMV RT kit (Promega). cDNAs were then purified on Centricon 30 (Amicon) and one-tenth of the resulting cDNA was used for amplification. PCR assays were performed in a Crocodile III cycler (Appligene) with the following amplification program: 94 °C for 5 min, 30 cycles of 94 °C for 30 s, 53 °C for 30 s and 72 °C for 30 s, and 72 °C for 10 min. The primers used were 5'HindIII55 (5' TTTAAATAAGCTTAGAACCAGCGGTTACCA 3') and 3'XbaI1220 (5' TTTAAATCTAGAAAGCAATAAAAACTGTACAATATTGGTC 3'). Five molecular clones were sequenced twice on both strands (see sequence analysis). The relevance of mutations was confirmed by PCR sequencing to determine consensus sequences.
Cell clones.
U373 cells were co-transfected with pLTRLuc and the h-CXCR4 plasmid. Clones were selected with 500 µg/ml neomycin sulphate (Gibco-BRL). These cells were then co-transfected with the pPGK-CD4 and the pPGK-hygroR plasmids to give the CD4h-CXCR4U373LTRLuc cell lines. Clones were selected by using 0·15 mg/ml hygromycin B (Calbiochem).
U373 cells were transfected with LTRLuc-PGK-hygro plasmid to obtain U373LTRLuc cells and clones were selected by using 0·15 mg/ml hygromycin B. These cells were then transfected with the agm-CXCR4 plasmid to yield agm-CXCR4U373LTRLuc cells, which were selected by using 0·5 mg/ml G418 (Gibco-BRL). The pCMV-CD4-SV-puroR plasmid was then transfected into the agm-CXCR4U373LTRLuc cells to give CD4agm-CXCR4U373LTRLuc cells, which were selected with 0·5 mg/ml G418, 0·3 mg/ml hygromycin B and 0·3 µg/ml puromycin (Calbiochem).
Cell fusion assays.
Syncytium-formation assays were performed by using adherent or non-adherent cells infected with HIV-1 and adherent target cells. The indicator target cells contained transiently or stably transfected pLTRLuc reporter plasmid or stably transfected pLTRLacZ reporter plasmid. Cell ratios were usually 1:1 for adherent cell co-cultures and 1:2 to 1:5 for adherent/non-adherent cell co-cultures. Samples were analysed 816 h later, depending on cell fusion efficiency.
Reporter assays.
Analyses were performed by using two indicator reporter gene systems (lacZ or luciferase), depending on the indicator cell used for in situ or quantitative analysis. In situ analysis measuring ß-galactosidase activity used cells fixed in 0·5% glutaraldehyde and X-Gal assays were performed after incubation for 4 h at 37 °C or overnight at 4 °C (Dragic & Alizon, 1993 ). Blue-stained syncytia were scored under a binocular microscope. Quantitative analyses were made by using a LB9501 luminometer (EGG Wallace) (Sol et al., 1993
). Quantitative chloramphenicol redß-galactopyranoside (CPRG) (Boehringer Mannheim) assays were also performed as described previously (Dumonceaux et al., 1998
).
Blocking experiments.
SDF-1 (100800 nM; R & D Systems) was added to 2x104 CD4-positive indicator cells per well in a 96-well plate 30 min before adding HeLa cells chronically infected with m7NDK (2x104 cells per well). Cells were co-cultured in triplicate and fusion efficiency was analysed 8 h later by a quantitative CPRG or luciferase test.
Sequence analysis.
The env expression vector and the sequencing method used have been described previously (Dumonceaux et al., 1998 ). The primers used to sequence the agm-CXCR4 plasmid were 5'HindIII55, 3'XbaI1220, 5'422 (5' TGTCATCTACACAGTCAACCTCTACAGC 3'), 3'501 (5' TTGGCCTCTGACTGTTGGTGGCGTG 3'), 5'540 (5' GGCCAAGGAAGCTGTTGGCTG 3') and pcDNA3-specific primers 5'pcDNA3 (5' AAATTAATACGACTCACTATAGGGAGACCC 3') and 3'pcDNA3 (5' CAACTAGAAGGCACAGTCGAGCC 3').
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Results |
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Effect of mutations in the C2, V3 and C3 regions
The mutations previously described in the C2, V3 and C3 regions of env were used as a basis for the correlation of tropism in simian cells with genetic changes, using chimeric constructs of m5 and wt env genes. A chimera corresponding to the mutations in the V3 and C3 regions of the m5NDK env gene inserted into the wt env gene was expressed in HeLa cells. These cells were cultured with indicator cells and fused with CD4-positive HeLa cells or CD4-positive COS-7 cells (Fig. 3). In contrast, the reverse chimera allowed only fusion with CD4-positive HeLa cells. CD4-negative indicator cells did not form syncytia. We conclude that the C3 and V3 mutations in the m5NDK gp120 protein are responsible for and sufficient to allow entry of m5NDK into CD4-positive COS-7 cells.
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Cloning of agm-CXCR4
As h-CXCR4 is the m7NDK isolate receptor, we postulated that agm-CXCR4 could act as the receptor for m5 and m7NDK virus. We therefore performed RTPCR on COS-7 cell RNA with oligonucleotides based on the h-CXCR4 sequence to isolate the agm-CXCR4 cDNA. This clearly demonstrated that agm-CXCR4 is expressed in COS-7 cells. We next subcloned the cDNA into a eukaryotic expression vector and compared its sequence with that of h-CXCR4. We found that five amino acids were different (Fig. 4); three in the N-terminal extracellular region (M24
I24; N35
H35; K38
R38), one in the second intracellular loop (R146
K146) and one in the second extracellular loop (N176
S176). This last change corresponds to a potential N-glycosylation site in the h-CXCR4 protein.
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While we cannot exclude the possibility that another receptor(s) is used in COS-7 cells, no other known co-receptor is expressed in U373 cells. Therefore, chemokine blocking experiments strongly suggest that agm-CXCR4 is the main receptor used by m5 and m7NDK to enter COS-7 cells.
The amount of agm-CXCR4 and virus entry
We postulated that the wtNDK isolate fuses with U373 cells bearing h-CD4 and agm-CXCR4 but not with h-CD4COS-7 cells (which constitutively express agm-CXCR4), because of the relative agm-CXCR4 expression levels.
As no anti-agm-CXCR4 antibody was available and because anti-h-CXCR4 antibody did not recognize agm-CXCR4, we performed transient experiments to overexpress agm-CXCR4. If the amount of agm-CXCR4 indeed restricts wtNDK virus entry, the overexpression of this receptor in CD4-positive COS-7LTRLuc cells should allow fusion with CEM cells chronically infected with these viruses. We thus transiently co-transfected CD4-positive COS-7 cells with pLTRLuc and different amounts of agm-CXCR4-expressing plasmid and co-cultured them with CEM cells infected with either wtNDK and m5NDK viruses (Fig. 7). As expected, no trans-activation was observed when the reporter gene alone was transfected into CD4-positive COS-7 cells after co-culture with CEM cells infected with wtNDK. When indicator cell lines were co-cultured with CEM cells chronically infected by m5NDK virus, we observed a 39·8-fold trans-activation of the luciferase gene. We did not observe a significant change in the degree of trans-activation when the amount of agm-CXCR4 was increased. However, when the co-culture was done with CEM cells chronically infected with wtNDK, we observed that the luciferase trans-activation level was directly correlated with the amount of agm-CXCR4 plasmid transfected, until a maximal level was attained (0·4 µg CXCR4) (Fig. 7). These results clearly demonstrate that CEM cells chronically infected with m5NDK virus fuse efficiently with CD4-positive COS-7 cells independently of agm-CXCR4 expression, whereas fusion mediated by wtNDK virus needs to pass a threshold level of agm-CXCR4 expression to become efficient. The amount of agm-CXCR4 surface expression is thus an essential parameter that restricts wtNDK virus entry.
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Discussion |
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We also demonstrated the importance of the C2 mutations for the CD4-independent entry of virus into COS-7 cells (Fig. 3). Indeed, we show here that the m7NDK virus can enter COS-7 cells in a CD4-independent manner. This result was expected, as we had previously found that m7NDK can enter cells independently of CD4 (Fig. 1
). Analysis of env genes from cells infected with either wt, m5 or m7NDK strongly suggests that the viruses have undergone a gradual genetic drift. Since the m5NDK virus has five of the seven mutations observed in the m7NDK virus, the appearance of these mutations seems to correlate with an extension of the entry spectrum.
If m5 and m7NDK viruses use h-CXCR4 to enter human cells, they could use a simian equivalent to enter COS-7 cells. We checked this by cloning agm-CXCR4 after RTPCR performed on COS-7 cell RNA (Fig. 4). We showed that the expression of this receptor was sufficient for m7NDK virus to enter non-permissive U373-MG cells (Fig. 5
). Experiments with SDF-1 showed that only a high concentration of the chemokine (800 nM) blocked the fusion between HeLa cells infected with m7NDK viruses and CD4-negative (data not shown) or -positive COS-7 cells (Fig. 6
). Nevertheless, as the results were similar for CD4-negative (data not shown) and-positive agm-CXCR4U373 cells (where no other receptor is known to be expressed), these results strongly suggest that agm-CXCR4 is the main receptor used by the mNDK viruses to enter COS-7 cells.
In the same way, CEM cells infected with wtNDK virus can fuse with h-CD4agm-CXCR4U373 cells but not with h-CD4COS-7 cells (Fig. 5). We postulated that there was more CXCR4 expressed in agm-CXCR4U373 cells than in COS-7 cells. This overproduction could not be demonstrated by FACS analysis, as all the antibodies we tested (12G5, Mab171, Mab172 and Mab173; R & D Systems) did not recognize agm-CXCR4 (data not shown) and because no other specific antibody is available at present. The importance of CXCR4 overexpression was thus confirmed by transient transfection of agm-CXCR4 plasmid in CD4COS-7 cells. The use of 0·4 µg agm-CXCR4 was sufficient to allow formation of syncytia with CEM cells infected with wtNDK virus, while untransfected cells did not form syncytia (Fig. 7
). When we increased the amount of agm-CXCR4 transfected, there was no further significant change in the efficiency of fusion observed. These results indicate that CEM cells infected with wtNDK virus cannot fuse with COS-7 cells when there is a normal expression level of agm-CXCR4, whereas CEM cells infected with m5NDK isolate fuse efficiently under the same conditions. The m5NDK virus enters COS-7 cells once a threshold level of agm-CXCR4 expression is reached, whereas wtNDK entry into COS-7 cells requires an increase in agm-CXCR4 expression level to be efficient. These results complement the findings of Platt et al. (1998)
, who showed that infection of cultured cells by R5-tropic HIV-1 strains is extremely sensitive to the level of CD4 expression that allows efficient interactions with virus gp120s. Furthermore, previous studies have demonstrated that infection of HeLa-CD4 clones by T cell-adapted NL4-3 is independent of CD4 content only once a threshold expression level is reached (Kozak et al., 1997
). Moreover, results obtained for CCR3 (Rucker et al., 1997
) have shown that this receptor serves as a functional co-receptor for a wider spectrum of viruses when it is present at a high concentration. Taken together, these results indicate that the entry of HIV-1 depends greatly on the affinity of the envelope protein for its receptors (CD4, CXCR4) but also on the level of co-receptor expression. Nevertheless, the overproduction of a co-receptor does not allow entry of all HIV isolates; wtNDK virus cannot infect CD4-positive cells that transiently express rat CXCR4 (Pleskoff et al., 1997a
).
Our results support the notion that, although the concentration of co-receptor is a major determinant of virus entry (Moore, 1997 ), some mutations in the env gene can overcome this restriction. The m5NDK isolate is thus the first example of a virus isolate in which a few mutations in the env gene can circumvent this hindrance to virus entry.
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Acknowledgments |
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References |
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Received 15 March 1999;
accepted 7 May 1999.