Bernhard Nocht Institute for Tropical Medicine, Bernhard Nocht Strasse 75, 20359 Hamburg, Germany, 2Heinrich Pette Institute, Martinistrasse 52, 20251 Hamburg, Germany, and 3Institute for Organic Chemistry University of Hamburg, Martin-Luther-King-Pl. 6, 20146 Hamburg, Germany
Received on April 4, 2000; revised on July 27, 2000; accepted on August 2, 2000.
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Abstract |
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Key words: HIV-1/infectivity/V3 loop/glycosylation/coreceptor
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Introduction |
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The gp120 glycoprotein is extensively glycosylated, and more than 50% of the molecular mass are carbohydrates (Leonard et al., 1990). Recently, it was shown in the SIV monkey model that SIVmac239 mutants lacking N-linked glycosylation within the V1 loop are more immunogenic compared to the SIVmac239 wild type (Reitter et al., 1998
). Highly reactive humoral immune responses were obtained after infection of monkeys with SIV strains containing such partially nonglycosylated external glycoproteins. In the same infected monkeys viremia of non-glycosylated mutants was low, but during the course of infection viruses resistant to neutralization by antibodies emerged. The resistant viruses were becoming predominant because a mutation had reintroduced the glycosylation site for N-linked carbohydrates in the SIV V1 loop. Thus, in the SIV model, the V1 loop carbohydrates are triggering the escape from V1-specific neutralizing antibodies. These data demonstrate that carbohydrates are responsible for masking immunodominant epitopes on the external glycoprotein and in this way carbohydrates can protect viruses against neutralizing antibodies.
Carbohydrates are also playing a role in the immune response against HIV-1 by masking the immunodominant V3 loop epitope (Schonning et al., 1996a). Resistance to or escape from V3 loop neutralizing monoclonal antibodies is usually seen by changes of the amino acid sequence in the antibody binding site, but it was also shown that HIV-1 escape from neutralization is caused by acquisition of carbohydrates (Schonning et al., 1996b
). Since the HIV-1 V3 loop is the major target for neutralizing antibodies (Rusche et al., 1988
), its masking by carbohydrates may influence the antigenicity and immunogenicity of total gp120.
Besides the role of glycosylation as a barrier for antibodies (Back et al., 1994; Cheng-Mayer et al., 1999
; Reitter et al., 1998
; Schonning et al., 1996a
,b), the V3 loop is the gp120 determinant that facilitates binding to the coreceptor molecules on CD4+ cells (Cocchi et al., 1996
; Kwong et al., 2000
). Recent data show that V3 peptides representing sequences of T-tropic strains directly bind to CXCR4 (Sakaida et al., 1998
). In addition to these experiments it was shown that enzymatically treated, nonglycosylated forms of HIV-1 (SF-2) gp120 bound much more readily to CXCR4 compared to the SF-2 gp120 containing intact carbohydrate structures (Bandres et al., 1998
). Thus, the totally nonglycosylated gp120 might present a more open coreceptor binding domain allowing a much better binding to coreceptors, an important step in HIV infection.
HIV strains are functionally classified with respect to their ability to use one or various coreceptor molecules for cell entry. Monotropic viruses, using only one coreceptor like CCR5 or CXCR4, are called R5 or X4 strains whereas dualtropic viruses, using either CCR5 or CXCR4, are called R5X4. In HIV transmission R5 strains are predominant and persist throughout infection (Rodrigo, 1997). In the late stage of HIV infection R5X4 and X4 strains have been shown to emerge (Kuiken et al., 1992
; Goudsmit, 1995
). Of clinical importance is the observation that the emergence of CXCR4-specific viruses correlates with the decline of CD4+ T-cell numbers and the progression to AIDS (Connor et al., 1997
; Spijkerman et al., 1998
). Mutations in the gp120 V3 loop can lead to a switch from the R5 to the X4 phenotype, which is a more pathogenic type of virus (Saag et al., 1994
; Goudsmit, 1995
).
To study the role of the V3 loop carbohydrates in CXCR4 specific infection, we have constructed a set of HIV-1 (NL4-3) molecular clones, all differing in the number of N-linked glycosylation sites within and around the V3 loop region. These V3 loop glycosylation mutants were tested for CXCR4-specific infection compared to the NL4-3 wild type. The overall conclusion of the present study is that NL4-3 viruses lacking two of the five V3 loop carbohydrate structures show enhanced infectivity and enhanced resistance to SDF-1, probably because the coreceptor binding region of these viruses is more exposed and more accessible to CXCR4 compared to viruses containing carbohydrate shielded V3 loop regions.
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Results |
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Our data demonstrate that infectivity of NL4-3 mutants for cells expressing high or low surface levels of CXCR4 or in the presence of SDF-1 appears to depend highly on the presence of g15 and g17, indicating that glycosylation of these sites affects the binding affinity for the viral receptors.
Modeling of the glycosylated V3 loop
A three-dimensional model of the V3 loop and the five carbohydrates structures was developed based on NMR structures of unglycosylated and glycosylated V3 loops as well as the information from the x-ray structure (Wyatt et al., 1998). Contrary to literature data that postulates that sequences NCT (g14) or NCS (g16) are not normally glycosylated the x-ray structure analysis of the truncated gp120/CD4/mAB complex showed unequivocally that this sequence as well as the CNIS sequence at the C-terminal end of the V3-loop carry carbohydrate chains. Using this information we performed a homology replacement of the amino acids of a synthetic glycosylated V3 loop to arrive at the sequence of NL4-3. The 3D structure of the glycosylated V3 loop was determined by NMR spectroscopy (Meyer et al., unpublished observations). The mutated V3-sequence was optimized by a MD simulation in a water box. The biantennary decasaccharide was chosen as a minimal structure to represent complex type sugars at g15. The oligomannosidic structures at positions g13, g14, g16, and g17 were represented by a Man8 structure. The 3D structure of these oligosaccharides was optimized and the local minima obtained were used to connect the oligosaccharides to the peptide backbone. Subsequently, a MD simulation of the resultant glycoprotein in a water box for 600 ps yielded the structure depicted in Figure 4a,b. Here, the carbohydrate chains largely shield the peptide from direct interaction with receptors on human cells.
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Discussion |
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Generating mutants differing in V3 glycosylation is based on amino acid exchanges altering the recognition site for N-linked glycosylation. In our study we have changed codons for g13 and g17 in such a way that the Asn codon was replaced by Gln, the chemically most similar amino acid. Changing the codon for g15 is more critical because changes in the sequence of the V3 loop can also directly influence the viral phenotype, which is mostly found if positively charged amino acids are introduced (De Jong et al., 1992). Alteration of the NNT recognition site for g15 by an exchange of Asn against Gln introduces an amino acid normally not present in V3 sequences (Korber et al., 1998
). Therefore, NNNT was exchanged for GSTT, NNNK, and NNNI, respectively, to produce sequences at this site that are also naturally occurring (Schreiber et al., 1994
).
In addition to amino acid variations of the gp120 V3 loop the oligosaccharide part is also highly diverse (Feizi and Larkin, 1990; Liedtke et al., 1997
). Both complex type and high mannose type structures are found depending on the expression system and the genetic sequence. To produce virus mutants containing the identical glycosylation pattern as present in vivo we therefore have used human PBMC instead of laboratory cell lines. Virus produced in PBMC cell cultures has the identical pattern of gp120 glycosylation as it is present in the in vivo situation. However, it has been established that the glycosylation pattern at an individual glycosylation site can also vary dependent on the presence or absence of other sites (Pfeiffer et al., 1994
). Thus, it is conceivable that complex type chains are replaced by high mannose carbohydrates at other sites outside the V3 loop or vice versa. Changing a complex type sugar to high mannose type can, for example, lead to the binding of MBP (mannose binding protein) that might influence viral infectivity (Ezekowitz et al., 1989
; Larkin et al., 1989
). However, each carbohydrate structure is a substantial space-occupying part of gp120 that is displayed on the outer surface of the virions able to interact with other molecules. A fundamental role of V3 based carbohydrates, especially at g15, is to shield the V3 loop from antibody binding. On the other hand, the V3 loop is interacting with the coreceptors CCR5 or CXCR4. Thus, prevention of antibody binding by carbohydrate shielding might also prevent the effective binding to the chemokine receptor molecules. The role of carbohydrates in shielding the V3 loop might become more complex since the coreceptors CCR5 and CXCR4 are differing in their carbohydrate content and future work is needed to study the role of V3 based carbohydrates for coreceptor switching.
A study defining the role of V3 loop carbohydrates was carried out by Nakayama et al. (1998). They also studied the role of V3 loop sugars using a mutant of NL4-3 lacking glycosylation at g15 (NNT to NNI). They reported that NL4-3 V3 glycosylation at g15 has a negative effect on infection of CXCR4 positive cells. In our experiments we have also shown that NNI has a negative effect that reduces viral replication by 30%. Another mutant lacking g15, the NNK mutant, containing a positively charged Lys instead of the Thr again showed a replication reduced by 30%. Both experiments demonstrate that lack of g15 alone had no dramatic effect on replication rate of the NL4-3 virus. Also, introduction of the positively charged amino acid had no enhancing effect on viral replication or infectivity. The GSTT mutant only lacking g15 showed a 3-fold increase in infectivity and furthermore this virus was able to infect the parental GHOST cells expressing low levels of CXCR4. This was only seen with the GSTT g15 mutant and not with the NNI nor NNK mutants. Therefore, it might be possible that the amino acid motif GSTT together with the lack of g15 is responsible for the enhanced infectivity observed. Losman et al. (1999)
also studied the influence of carbohydrates at g15 on CXCR4-specific infection. Using a mutant of the HIV-1 strain BRU lacking g15, they showed that the lack of a single carbohydrate structure neither impairs nor improves infectivity. In our study, the same weak effects on viral replication were observed with viruses lacking g13 or g17. Thus, in all studies, only little or no effects on CXCR4-specific infection were observed using mutants lacking a single V3-based carbohydrate structure.
To further test the effect of carbohydrates on infectivity, we have tested all permutations of doubly and triply deglycosylated virus mutants (Figure 1). Our data demonstrate that mutants lacking g15 and g17 showed a dramatic increase in infectivity (Table I). The enhancement of infectivity was only seen with the g15/g17 double and g13/g15/g17 triple mutants of 7-fold and 12-fold, respectively. Interestingly, the lack of g15 in combination with g13 did not show any effect on infectivity. Therefore, our data indicate that carbohydrates at g15 and g17 are responsible for an efficient shielding of the V3 loop (Kwong et al., 2000). Double mutants lacking g15 and g17 showed enhanced infection, were able to use low CXCR4 levels, and were highly resistant against SDF-1. Fully glycosylated NL4-3 and all other glycosylation mutants did not show such CXCR4-specific properties. Because of these reasons we suggest that both carbohydrates at g15 and g17 are responsible for the masking of the CXCR4 binding region of gp120.
Using a three-dimensional model of the V3 loop the role of g15 and g17 for masking the V3 loop was visualized. Both carbohydrates are close to the tip of the V3 loop and are together covering the GPGRAF motif which is the conserved part of the V3 loop of B subtype viruses. This GPGRAF motif as well as the left side of the loop are the targets for broadly neutralizing antibodies. Since both carbohydrates are close to these regions it is suggestive that masking by g15 and g17 can prevent the binding of neutralizing antibodies against the V3 loop. On the other hand, masking by carbohydrates might reduce the affinity not only for neutralizing V3-antibodies it might also reduce the binding affinity for the CXCR4 coreceptor.
It has been reported that the carbohydrate at g15 is responsible for shielding SIV or HIV-1 against immune recognition and against binding to neutralizing antibodies (Reitter et al., 1998; Schonning et al., 1996a
,b). In the SHIV model it was shown that escape of SHIV SF33 from neutralizing immune response was due to the reintroduction of the g15 glycosylation site (Cheng-Mayer et al., 1999
). In agreement with our study and the observations of Nakayama et al. (1998)
and Losman et al. (1999)
, lack of a single SF33 glycosylation site at g15 showed no dramatic effect on viral replication. Taken together these data indicate that the coreceptor binding site of X4 viruses is only slightly affected by alterations at g15 alone. To make this domain more accessible to CXCR4 the lack of a second glycosylation site downstream of g15 seems to be relevant. From the 3D model of the gp120 trimer made by Moulard et al. (2000)
and Kwong et al. (2000)
it seems to be clear that the V3 loop is the most important part that influences binding to CXCR4. In addition to their data on the influence of amino acid exchanges on the binding specificities of the gp120 trimer our data demonstrate a role for g15 together with g17 in masking the coreceptor binding surface of the gp120 trimer.
It has been reported that carbohydrates of gp120 can mediate binding of viruses to the host cells by an interaction of high mannose glycans with mannose receptors (Ezekowitz et al., 1989; Larkin et al., 1989
) or by a galactose specific interaction of the terminal residues of complex type chains (Manca, 1992
). The change of complex type sugar to a high mannose or hybrid type structure would also change the nature of the mannose and galactose mediated interactions and might thus contribute to the infectivity. Besides studies on HIV, a role for carbohydrates in modulating virusreceptor interactions was demonstrated for influenza A. It has been shown that two carbohydrate moieties in the vicinity of the receptor binding site of the hemagglutinin (HA) are regulating the receptor binding affinity. Lack of the sugars attached to Asn 123 and Asn 149 results in strong binding of HA to neuraminic acid-containing receptors. Viruses lacking both sugars were unable to elute from cells carrying receptors but binding was abolished in the presence of the two HA sugars in the sialylated form (Ohuchi et al., 1997
). Thus, the biological activity of HA, the binding to and dissociate from its receptor, is regulated by two carbohydrates. These observations demonstrate that besides the various functions of carbohydrates, like the stabilization of protein conformations and the protection against proteolytic degradation, sugars have direct effect on the affinity of, for example, viral glycoproteins to their cellular receptors.
In summary, our studies on genetically engineered NL4-3 mutants demonstrate that carbohydrates within and around the V3 loop are shielding the CXCR4 binding region of HIV-1. The loss of two carbohydrates increases CXCR4-specific infectivity, which is a competitive advantage against other virus mutants. This becomes important during progression of HIV infection to AIDS when the virus faces a drastically compromised immune system. Normally, these mutants are eliminated by the immune response because the deglycosylated V3 loop is much more accessible to neutralizing antibodies. In fact, such mutants can be detected as dominant species in AIDS patients (Schreiber et al., unpublished observations).
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Materials and methods |
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Cloning of V3 loop mutants
To replace the BglIINheI 225 bp fragment containing the V3 region we synthesized two oligonucleotides of 144 bases length with a 31 base pair overlap (pNL4-3 env positions 7016 to 7273). Oligonucleotides were hybridized and PCR amplified using BglII and NheI primers (BglII: 5'- GTA ATT AGA TCT GCC AAT TTC ACA GAC; NheI: 5'-TAA TTT GCT AGC TAT CTG TTT TAA AGT G). Glycosylation sites g13 and g17 (see Figure 1), were mutated by changing the codon for Asn (AAC) into the codon for Gln (CAA) (Reitter et al., 1998). Glycosylation site g15 was mutated by changing the 3 codons for Asn (AACAACAAT) into GGATCTACA, encoding Gly-Ser-Thr, a mutated glycosylation site identified in V3 sequences of patient isolates (Schreiber et al., 1994
). Using chemical DNA synthesis and PCR we generated four DNA fragments lacking the glycosylation sites g13/15, g13/17, g15/17, and g13/g15/g17. The sites g13 and g15 are located within the BglIIXbaI fragment whereas site g17 is located within the XbaINheI fragment. To generate all possible eight combinations of the three glycosylation sites, the four remaining clones were generated by crossing BglIIXbaI and XbaINheI fragments. To generate the respective proviral plasmids, BstEIIBamHI fragments were isolated from the pUCenv constructs and were cloned into the BstEII and BamHI sites of the pNL4-3-Bst vector (Figure 1).
Transfection of cells
Vector pNL4-3-Bst DNA of the respective glycosylation mutant was isolated from XL-1 bacteria by standard procedure (Qiagen, Endo Free Plasmid Maxi Kit). DNA was transfected into Hela cells using the calcium phosphate technique (Chen and Okayama, 1987). Hela supernatants were used for the infection of IL-2 and PHA stimulated PBMC.
Cell culture
Hela-P4 cells were grown in Dulbeccos modified Eagle medium (DMEM) (Life Technologies, Karlsruhe, Germany) containing 10% fetal calf serum (FCS) (Biochrom KG, Berlin, Germany). PBMC were obtained from HIV negative donors and isolated by Ficoll-Paque gradient centrifugation. PBMC were prestimulated with 5 µg/ml phytohemagglutinin (both from Biochrom KG, Berlin, Germany) and then cultivated in RPMI medium containing 10% fetal calf serum, and 100 U/ml of interleukin 2 (Chiron GmbH, Ratingen, Germany). Stimulated PBMC were infected with Hela cell culture supernatants and virus production was measured by a standard p24 assay (Moore et al., 1990). To test the coreceptor usage human osteosarcoma (GHOST) indicator cell lines (provided by D.Littman) were cultured in DMEM supplemented with 10% fetal calf serum. The parental GHOST cell clone expressing human CD4 was grown under G418 selection (500 µg/ml; Life Technologies GmbH, Karlsruhe, Germany). GHOST cells expressing one of the coreceptors CCR3, CCR5, CCR8, BOB, Bonzo, or CXCR4 were cultivated in medium additionally supplemented with puromycin (1 µg/ml; Sigma, Deisenhofen, Germany).
Evaluation of coreceptor usage
The p24 immunostaining of infected cells was performed as described earlier (Chesebro and Wehrly, 1988; Clapham et al., 1992
). In brief, infected cells were grown in Dulbeccos modified medium containing 10% fetal calf serum for 4 days, washed in phosphate-buffered saline (PBS), and fixed using methanol-acetone (20°C, 1:1). For p24 detection a mixture of two mouse monoclonal anti-p24 antibodies (EVA365, EVA366, provided by the MRC AIDS reagent project) was applied at a dilution of 1:400. Bound antibody was detected using goat anti-mouse IgG antibodies conjugated to
-galactosidase (1:100 dilution, Biozol, Germany). Staining was performed using 0.5 mg x-gal (5-bromo-4-chloro-3-
-D-galactosidase) per ml PBS containing 3 mM potassium ferrocyanide, 3 mM potassium ferricyanide, and 1 mM magnesium chloride for 12 h at room temperature. Stained blue cell clusters were counted and expressed as foci forming units (ffu). Infection was blocked by adding SDF-1.
Flow cytometry
GHOST indicator cells were stained with anti-CXCR4 monoclonal antibody conjugated to R-phycoerythrin (RPE, Pharmigen, Hamburg, Germany). Cells were incubated with anti-CXCR4-RPE antibody for 15 min at room temperature in 100 µl phosphate-buffered saline (PBS) containing 1% FCS. Cells were washed twice in PBS-1% FCS and twice in PBS, and were resuspended in PBS (1 x 106 cells/500 µl). Fluorescence was measured on a FACSCalibur (Beckton Dickinson).
SDF-1 inhibition
GHOST-CXCR4 cells were incubated with SDF-1 (Strathmann Biotech, Hannover, Germany) 2 h prior to infection. Virus supernatants from IL-2 and PHA stimulated PBMC of NL4-3 mutants containing infectious virions equivalent to 60100 ffu were used for infection. Cells were washed on day 2 and immunostaining was performed as described above on day 4. Infection of GHOST-CXCR4 cells was expressed as ffu per ml or ffu per ng of p24.
Patient isolates were grown in IL-2 and PHA stimulated PBMC. Virus supernatants containing 50 TCID50 were applied to IL-2 and PHA stimulated HIV-1 negative donor PBMC together with SDF-1. Cells were incubated 18 h at 37°C, washed four times in RPMI 1640. Infection was monitored by standard p24 assay (29) (Aalto Bio Reagents Ltd., Dublin, Ireland).
Three-dimensional model generation
The structures shown in Figure 4 were obtained by homology modeling based on V3 loop NMR data published by Gupta et al. (1993) and a glycosylated V3 loop by Meyer et al. (unpublished observations). Modeling and visualization was performed on Silicon Graphics Octane computers using the SYBYL software package. The 3D structure of the individual carbohydrate structures was optimized using the GEGOP program (Stuike-Prill and Meyer, 1990
). These oligosaccharide structures were then attached to the peptide and subjected a 600 ps MD simulation in a water box. The resulting relaxed structure is shown in Figure 4a,b.
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Acknowledgments |
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Abbreviations |
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Footnotes |
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References |
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