www.elsevier.com/locate/yviro

Virology 324 (2004) 90–102

HIV-1 acute infection env glycomutants designed from 3D model:

effects on processing, antigenicity, and neutralization sensitivity

Frédéric Reynard, Ahmed Fatmi, Bernard Verrier, and Frédéric Bedin*

FRE 2736 CNRS-bioMérieux, IFR128 Biosciences, CERVI, Lyon, France

Received 9 February 2004; returned to author for revision 5 March 2004; accepted 22 March 2004

Abstract

The human immunodeficiency virus type 1 (HIV-1) envelope protein (Env) has evolved to limit its overall immunogenicity by extensive

glycosylation. Only a few studies dealing with glycosylation sites have taken into account available 3D data in a global approach. We

compared primary env sequences from patients with acute HIV-1 infection. Conserved N-glycosylation sites were placed on the gp120-3D

model. Based on vicinity, we defined glycosylation clusters. According to these clusters, we engineered plasmids encoding deglycosylated

gp160 mutants. We also constructed mutants corresponding to nonclustered glycans or to the full deglycosylation of the V1 or V2 loop. After

in vitro expression, mutants were tested for functionality. We also compared the inhibition of pseudotyped particles infection by human-

neutralizing sera. Generally, clustered and nonclustered mutants were affected similarly. Silencing of more than one glycan had deleterious

effects, independently of the type of sugar removed. However, some mutants were moderately affected by glycans removal suggesting a

distinct role for these N-glycans. Additionally, compared to the wild-type pseudovirus, two of these mutants were neutralized at higher sera

dilutions strengthening the importance of the location of specific N-glycans in limiting the neutralizing response. These results could guide

the selection of env mutants with the fewest antigenic and functional alterations but with enhanced neutralization sensitivity.

D 2004 Elsevier Inc. All rights reserved.

Keywords: HIV-1; Env glycoprotein; N-glycans; Neutralization; Env processing; Acute infection

Introduction

The envelope glycoprotein (Env) of human immunode-

ficiency virus type 1 (HIV-1) promotes binding of virus to

target cells through CD4/chemokine receptors complex and

triggers events leading to fusion between viral and cellular

membranes.

During and after translation, the envelope precursor

gp160 folds in endoplasmic reticulum and acquires the

ability to oligomerize before being exported to the Golgi

apparatus. In the Golgi, gp160 is proteolytically cleaved to

create two noncovalently associated subunits, the gp120

(surface protein) and gp41 (transmembrane protein). The

gp120/gp41 association is labile and may dissociate (or

‘‘shed’’) spontaneously (McKeating et al., 1991). The

0042-6822/$ - see front matter D 2004 Elsevier Inc. All rights reserved.

doi:10.1016/j.virol.2004.03.022

* Corresponding author. FRE 2736 CNRS-bioMérieux, CERVI, 21

Avenue Tony Garnier, 69007 Lyon, France. Fax: +33-437-28-24-11.

E-mail address: bedin@cervi-lyon.inserm.fr (F. Bedin).

gp120 consists of a core that is mostly conserved among

different strains and highly variable surface-exposed loops.

Gp160 (about 850 residues) contains about 28 sites for

N-linked carbohydrates attachment. Nearly 50% of the

gp120 mass is due to carbohydrate residues and a broad

variety of both high-mannose and complex-type have been

identified (Leonard et al., 1990; Zhu et al., 2000). Such

extensive glycosylation is very unusual regarding to the

envelope glycoproteins of others viruses. As a comparison,

the F and H glycoproteins of measles virus have, respec-

tively, 3 and 5 glycosylation sites for 550 and 617 residues

(Cattaneo and Rose, 1993). Sugars play an important role in

structural folding of nascent proteins in endoplasmic retic-

ulum, in precursor cleavage in the early Golgi apparatus,

and consequently modulate the biological properties and the

infectivity of the virus (Benjouad et al., 1992; Fischer et al.,

1996). Nevertheless, N-glycans appear to be dispensable for

maintaining gp120 native structure once formed (Fennie and

Lasky, 1989; Li et al., 1993). Glycosylation is also a mean

of diversifying proteins without recourse of the genome and

a growing body of evidence demonstrates that it modulates

F. Reynard et al. / Virology 324 (2004) 90–102 91

the exposure of the Env protein to immune surveillance in

patients or in experimentally infected animals, acting like a

shield (Cheng-Mayer et al., 1999; Schonning et al., 1998;

Wei et al., 2003a, 2003b).

The envelope glycoprotein is the target of virus-neutral-

izing antibodies; however, it does not elicit efficient neu-

tralizing response in infected people. The high number of N-

glycans could partially explain why broad neutralizing

antibodies directed against envelope glycoprotein have not

been frequently identified.

Several reports have described individual substitutions of

N-glycosylation sites located on domains implied in the

resistance to antibody-mediated neutralization. These

domains include V1/V2 or V3 loops that mask CD4 binding

sites or epitopes induced after binding of the CD4 (CD4-

induced epitopes). Except for the gp41 moiety and for the

gp120 variable loops, few systematic has been made,

especially regarding the spatial location of N-glycans (John-

son et al., 2001; Desrosiers et al., 1998; Hemming et al.,

1996; Lee et al., 1992; Ogert et al., 2001). In addition,

studies were mainly conducted on SIV or HIV-1 T-cell line-

adapted viruses (TCLA). It is now well accepted that this

type of virus is antigenically faraway from primary clinical

isolates and typically more sensitive to antibodies neutral-

ization (Moore et al., 1995).

In order to be as close as possible to the virus that has

founded the infection, we chose to base our study on gp120

sequences from HIV-1 acute infection primary isolates. It

was recently postulated that the ‘‘glycan shield’’ was evolv-

ing during the course of infection in the face of a changing

antibody repertoire (Wei et al., 2003a). We made the

hypothesis that at the time of acute infection, the well-

conserved glycosylation sites would be located in specific

regions at the surface of the protein and serve to protect

against humoral immune response gp120 epitopes critical

for HIV-1 transmission. According to the 3D-model data,

we mutated potential glycosylation sites to remove sugars

on these different regions.

In this approach, the major obstacle was to increase

immunogenicity without altering the oligomeric gp120:

we monitored the effects on Env processing and function;

we also tested the neutralizing activity of human sera on

pseudotyped particles harboring mutated envelopes. These

data could be useful for rationalizing the design of new Env

immunogens that would induce a strong broad range anti-

body response against the HIV-1.

Results

Rational of deglycosylation constructs

To identify conserved N-linked glycosylation sites of

HIV-1 gp120, 14 primary env sequences obtained during

acute infection and before seroconversion were aligned

(Fig. 1). These sequences were generated from patients

infected sexually (eight homosexuals, six heterosexuals).

The sequence alignment revealed the presence of 10 well-

conserved N-glycosylation sites (defined by the NX{-P}T/

S sequon), spread all over the gp120 amino acids sequen-

ces (N44, N88, N109, N123, N148, N201, N243, N253,

N289, and N301/307). The N185 site was present in all

sequences from homosexually transmitted isolates. Two

other potential sites (N136, N142) were conserved among

env sequences from heterosexually transmitted isolates. The

133 env sequence retained for mutants construction has

been isolated from a patient contaminated by the homo-

sexual route; consequently, the N185 site was included in

the study. Due to the systematic presence of at least one

sugar at the close positions N231/N237, the N231 site was

also included. The N136/N142 sites, as well as the N81

site, were not considered as they were absent from the 133

env sequence. Due to their proximity on 133 sequence, the

N-glycan sites N180/N185 (as N201/N208) are removed

simultaneously.

To analyze the 3D distribution of the selected N-glyco-

sylation sites on the 133 sequence, we used the gp120

model based on the X-ray crystallography data (Kwong et

al., 1998). Each potential site was highlighted on the 3D

model. The selected sites were arbitrarily grouped into three

nonoverlapping ‘‘regions’’ (or ‘‘cluster’’) based both on the

proximity of each site with the others (Fig. 2). The size of

each cluster was roughly delimited by a 20 Å circular area,

considered as the typical antibody footprint (Pantophlet et

al., 2003). The first region (colored in red; A region; Fig.

2A), constituted by sites N44, N231, and N243, was located

approximately on the bridging sheet, which is implied in

receptor and coreceptor binding. The second region, pre-

sented in blue (B region; Fig. 2B), grouped the positions

N148, N180/N185, N201/N208. This cluster was separated

from the first one by several residues (Q227, L262, and

P279) critical for coreceptor binding (Rizzuto et al., 1998).

Two out of four glycosylation sites in the B cluster (N180

and N185) also belonged to the 2G12 neutralization epitope

(Scanlan et al., 2002). The third region (C region; Fig. 2C),

represented in yellow (N109, N253 and N289), was located

on the gp120 silent face, which is well exposed at the

surface of the envelope oligomer but does not induce

antibodies. Among the remaining positions, the N88 and

N301/307 sites were not retained for our study, as they

belonged to the inner domain of the gp120 that faces up the

gp41 moiety. This domain is poorly accessible in Env

oligomers and does not induce neutralizing antibodies. As

the N123 site (colored in green) remained alone, it was

grouped with sites in the neighborhood at positions N109

and N289 (C cluster) or N148 (B cluster) and defined the

‘‘D’’ mutants. All of four were roughly located near the

base of the V3 loop. These mutants were considered as a

separate group.

The clusters definition was validated by checking con-

served N-glycans location on 3D model for the 13 others

primary gp120 sequences (data not shown).

Fig. 1. Positions of the potential glycosylation sites on 14 gp120 primary sequences from patients with HIV-1 acute infection. The amino acids numbering of N

mutations corresponds to the HIV-1HXB2 gp120 sequence deleted for a part of C1 and C5 regions and for the V1V2 loop as described by Kwong et al. (1998).

The code number of the patients and the contamination route, homosexual (HM) or heterosexual (HT), identify each sequence. The position of the missing

V1V2 loop is indicated by a triangle.

F. Reynard et al. / Virology 324 (2004) 90–10292

For each region, several deglycosylation mutants were

engineered (Table 1); in a series of mutants, all the N-

glycan attachment sites belonging to the same region were

removed (g4A, g5B/g6B, g5C, respectively, for the A, B,

and C clusters). As several intermediates were generated

for this purpose, they were also included in the study

(g1A–g3A, g1B–g4B, g1C–g4C). In parallel, the effects

induced by two (g1), three (g2), or four mutations (g3),

but not located in the same region, were evaluated (Fig.

2D). The deglycosylation of the V1 loop (g4) or the V2

loop (g5) are modifications that improve the capacity of

the Env protein to induce neutralizing antibodies or its

sensitivity to neutralizing sera (Reitter and Desrosiers,

1998; Quinones-Kochs et al., 2002). We made these

mutations on the 133 env sequence to complete our study

because the crystallized core-gp120 is devoid of the V1/

V2 loops. For all the constructs, the potential N-glycosyl-

ation sites were removed by replacing the asparagine

residue (N) in the NX{-P}T/S sequons by a glutamine

residue. Glutamine was chosen because of its structural

similarity with asparagine differing by only a single

methylene group. In addition, a minimum of two nucleo-

tides changes are necessary to revert to the original

asparagine codon. All the constructs were checked by

sequencing both strands to exclude mutations induced by

PCR during mutagenesis.

Fig. 2. (A–C) Positions of the mutated N-glycosylation sites on the 133 gp120 sequence according to the gp120 crystallized model viewed on three different

angles. Each asparagine from the canonical motif NX{-P}T/S is highlighted by different colors according to its respective region. The amino acid numbering

corresponds to the variable loops deleted envelope sequence. The missing V1/V2 structure is schematized by a gray oval. The 20 Å bar represents

approximately the size of an antibody footprint. (D) Positions of asparagine residues according to the nonclustered mutants definition viewed on two different

angles.

F. Reynard et al. / Virology 324 (2004) 90–102 93

Influence of N-glycans removal on gp160 cleavage and

gp120 shedding

To determine the effect of cluster deglycosylation on

envelope processing and particularly on cleavage, 293T

cells were transfected and both the lysates and the cell

culture supernatants were analyzed by an anti-gp120 im-

munoblot (Fig. 3). Luminescence scanning was used to

detect the immunoreactive bands on immunoblots on cell

lysates. As the Rev protein is necessary to Env synthesis,

we checked that it was present at the same level in all

lysates (data not shown). All constructs were expressed at

a comparable order of magnitude (Fig. 3). The mobility of

the gp160 and gp120 gene products expressed from g1A–

g2A, g1B, g1C–g2C, and g1D constructs appeared to be

close to the one of the wild-type proteins. For these

mutants, only one or two glycosylation sites were re-

moved. The gp160 of all others mutants migrated slightly

faster than wild-type gp160. This result confirmed the loss

of glycans caused by mutagenesis; the glycosylation of one

N-glycan attachment site is predicted to contribute approx-

imately 2–3 kDa to the apparent molecular mass of a

protein.

For the mutants with one N-glycan removed (i.e., g1A,

g2A, g1B, g1C, g1D, but not g2C), gp120 was detected in

cell lysates. Gp120 was also detected for g3C (two muta-

tions), g4 (three mutations), and g3B (four mutations). For

g2B, g4B, g6B, g2C, g4C, g5C, and g2D, the gp120 was

exclusively detected in the culture supernatants: a particu-

larly important shedding caused absence of detectable

gp120 in cell lysates. An Env bearing one unique mutation

at N109 or N148 was affected similarly indicating the

leading role of these mutations in gp120/gp41 dissociation

(cf. g2C; data not shown for N148). For the remaining

mutants, no gp120 was detected both in the lysate and the

supernatant; for these mutants, the gp160 cleavage was

severely impaired. The removal of the three potential sites

present on the V1 loop (g4) had no effect on the cleavage

rate compared to the native protein. Cleavage on nonclus-

tered mutants was affected in the same way as the clustered

mutants. Additionally, the effects were apparently indepen-

dent of the nature of the removed sugars (complex or high-

mannose type, cf. Table 1).

Influence of N-glycans removal on cell membrane

presentation and incorporation in pseudovirion

Cell-to-cell fusion assays on GHOST-CCR5 cells were

performed (Fig. 4A). This type of experiment was carried

out with 293T cells expressing the different mutants. The

Table 1

Glycosylation status of the engineered mutants

Region Mutants Positions of

mutated N-glycan

attachment sites

gp120 regions

mutated

N-glycans

removed

WT no-mutation

wild type

– –

anti no-mutation

insert in reverse

orientation

– –

(A) Red g1A 44 C2 (V1V2 stem) 1C

g2A 243 V4 1C

g3A 243–231 V4–V4 1C1M

g4A 44–231–243 C2–V4–V4 2C1M

(B) Blue g1B 180/185 C3 2M

g2B 180/185–148 C3–V3 1C2M

g3B 180/185–201/208 C3–C3 1C2M1U

g4B 180/185–148–

201/208

C3–V3–C3 1C3M1U

g5B 180/185–201/

208–231

C3–C3–V4 1C3M1U

g6B 180/185–148–

201/208–243

C3–V3–C3–V4 2C3M1U

(C)Yellow g1C 289 C4 1M

g2C 109 C2 1H

g3C 289–253 C4–V4 1M1C

g4C 289–109 C4–C2 1M1H

g5C 289–253–109 C4–V4–C2 1M1C1H

(D) Green g1D 123 C2 1C

g2D 123–148 C2–V3 2C

g3D 289–109–123 C4–C2–C2 1C1M1H

Misc. g1 289–123 C4–C2 1C1M

g2 289–123–243 C4–C2–V4 2C1M

g3 289–123–243–

180/185

C4–C2–V4–C3 2C3M

g4 V1 deglycosylated V1 2C1U

g5 V2 deglycosylated V2 2C

For each mutant, location, number, and nature of sugar removed are

indicated. C: complex-type sugar, M: high-mannose type sugar, H: hybrid

type sugar, and U: unknown type sugar. The numbering of N-glycan

positions is according to Fig. 1.

F. Reynard et al. / Virology 324 (2004) 90–10294

fusion capacity was dependent on the correct processing of

the Env glycoprotein and on its exportation to the cellular

membrane. The number of syncytia decreased with the

number of mutations, correlating with the gp160 cleavage

impairment and edifice instability as described in previous

section. Compared to the wild-type Env, the loss of one or

Fig. 3. gp160 and gp120 immunoblots after 293T cells transfection. Detection was

weight standard of migration (kDa). To normalize sample loading, an anti-h-actinsupernatant.

sometimes two glycosylation sites (g3A, g3C, and g1) had

little impact on syncytia formation. This biological assay is

more sensitive than an immunoblot, and we noticed a fusion

capacity even for some mutants for which no gp120 was

detected on blot (i.e., g3A, g1 or g5, and in a lesser extent,

g5B and g2). No fusion was observed for g4A, g5C, g3D

(three mutations), g3 (five mutations), and g6B (6 muta-

tions). For g6B and g5C, the gp120 was detected by

Western blot in supernatant but not in cell lysate, suggesting

that the protein was exported to the cell surface and the

gp120 heavily shed. Apparently, the remaining gp120/gp41

was not able to induce cell-to-cell fusion. Unexpectedly, the

mutants g4 and g3B with three and four mutations, respec-

tively, had a fusion capacity comparable to the wt. Glyco-

sylations located near or within the V1/V2 or the V3

variable loops could play an important role in coreceptor

usage and CD4 dependence (Kolchinsky et al., 2001;

Pollakis et al., 2001). We tested fusion on CD4+–CXCR4+

cells and CD4�–CCR5+ cells (GHOST-CXCR4 and HOS-

CCR5, respectively). In our hands, no mutant proteins

induced fusion on these cells (data not shown).

The exportation of Env to the cell membrane does not

presume its incorporation into virions. In order to know if

the mutant proteins could be incorporated with preserved

functionality into viral particles, we generated pseudotyped

particles with the different mutants and tested the capacity

of these pseudoviruses to infect several GHOST cell lines

(Fig. 4B). The envelopes for which no or few cell-to-cell

fusion occurred showed no infection, neither on GHOST-

CCR5 nor on GHOST-CXCR4. The results for g4B, g2C,

g4C, g2D, and g5 were more ambiguous because no

infection was noticed although cell-to-cell fusion assay gave

syncytia. For mutants g3A and g1, the level of infection was

unexpectedly weak considering their important fusion ca-

pacity. These results were reproducible and indicated a

difference between Env presented to the cell surface and

Env incorporated in enveloped pseudovirions.

Immunoblots carried out on pseudoparticles after sedi-

mentation by ultracentrifugation on sucrose cushion con-

firmed the previous results (Fig. 4C); the gp120 and the

gp41 were detected only on pseudoviruses that infected

indicator cells the most efficiently. For g2C and g4C, the

gp41, but not the gp120, was detected.

made with a polyclonal antiserum directed against gp120. MW: molecular

monoclonal antibody was used (A; bottom). L: cell lysates; S: cell culture

Fig. 4. Env exportation to cell membrane, incorporation in pseudoparticles, and infectivity. (A) Cell-to-cell fusion assay. 293T cells expressing the different

mutant genes were cocultivated with GHOST-CCR5 cells. (� ): fusion as on negative control, that is, < 5 syncytia/10 fields; (++): fusion equivalent to wildtype, that is, 60–100 syncytia/10 fields; (+) and (+/� ): intermediate levels of fusion, that is, 20–50 and 5–15 syncytia/10 fields, respectively. Resultscorrespond to the average of two independent experiments conducted in duplicate. (B) GHOST cells infections by pseudoparticles. Pseudotyped particles were

incubated with GHOST-CCR5 cells and subsequent luminescence was read in cell lysates. Results correspond to the average of two experiments made in

triplicate. (C) Purified pseudoparticles immunoblotted with a polyclonal antibody directed against gp120 (top) and gp41(bottom). Samples were normalized

after p24 quantification.

F. Reynard et al. / Virology 324 (2004) 90–102 95

Influence of N-glycan removal on sCD4 binding

The ability of Env to bind CD4 is an important feature

that could be used to give information about the general

conformation and the functionality of the mutants. Antigen-

capture ELISAs were carried out on both cell lysates and

culture supernatants of transfected 293T cells (Fig. 5). The

samples were the same with those used for the Western

blots. These ELISA were revealed with soluble CD4

(sCD4)/anti-CD4 antibody. Mutants with important alter-

ations such as g4A, g5B, g3D, or g2–g3 have not been

taken into account. Compared with the gp120 detected in

lysates, the signal in culture supernatants reflected both the

cleavage efficiency and the instability of the gp120–gp41

association. These results confirmed Western blot experi-

ments (Fig. 3) but with more sensitivity. Taken as a whole,

the removal of several glycosylation sites had little impact

on sCD4 binding. However, for g6B-6 mutations (and in a

lesser extent, for g3B-4 mutations), the decrease in super-

natant signal was probably attributable to antigenicity

disturbance because the relative amount of gp120 detected

on blot was equivalent to gp120 detected for g1B or g4B

(Fig. 3). When ELISA with serial dilutions of sCD4 were

performed, some mutants with one mutation had a relative

affinity better than the wild-type protein; the sCD4 half-

maximum binding was 0.05 Ag/ml for mutants g2A, g1B,and g1C, and 0.15 Ag/ml for wild-type (data not shown).Removal of N243, N180/185, or N289 improved sCD4

binding.

In order to compare the respective effects of mutations on

both sCD4 binding and overall antigenicity, another ELISA

was carried out with the autologous serum from patient

#133 (Fig. 5). The results were comparable to those

obtained on the sCD4-ELISA. Mutations had little impact

on sera recognition compared to native protein. No signif-

icant difference was noticed between clustered and non-

clustered mutants. Surprisingly, although the g2C, g4C, g5C

mutants were relatively well recognized by serum, no signal

was monitored for sCD4 binding probably because muta-

tions affected mainly CD4 binding site. For g1 and g3A,

despite the lack of gp120 detected in Western blot (Fig. 3),

we found a little but significant signal in supernatant and

lysate indicating that these mutants were cleaved and

exportated.

Neutralizing activity against pseudoparticles bearing Env

glycomutants

To ascertain the influence of the glycosylation on sensi-

tivity to antibody-mediated neutralization, four sera speci-

men from HIV-1-infected patients were tested for their

ability to neutralize infectious pseudotyped particles in an

in vitro neutralization assay using GHOST CD4+–CCR5+

cell line. One serum was taken from patient #133 two years

after the acute phase, and the three others corresponded to

titrated neutralizing sera from a previous study (Burrer et al.,

2001). These sera had a measurable neutralizing activity

against several primary isolates. The results are displayed in

Fig. 5. Accessibility of the CD4 binding site. ELISAwas carried out both on lysates (A) and culture supernatants (B) from transient expression in 293T cells.

Detection was made with sCD4/anti-CD4 antibody and with the autologous serum from patient #133. Results correspond to the average of two independent

experiments conducted in duplicate. The threshold (horizontal line) was defined as 3 times the background read on the negative samples.

F. Reynard et al. / Virology 324 (2004) 90–10296

Table 2 as 50% inhibitory titer (IT50). The results were

compared to those obtained for the parental env gene. The

four neutralizing sera showed a greater inhibitory titer for

two glycomutants than for the wild type; CCR5-mediated

Table 2

Neutralization of pseudoviruses with 1:2 serial dilutions of neutralizing and

nonneutralizing human sera

Pseudovirions Neutralizing sera

1 2 3 4 5

g2A 570 180 60 1300 20

g1B 820 150 60 1300 80

g3B 1350 1120 250 1600 80

g1C 510 120 45 400 80

g3C 490 150 25 800 50

g1D 640 160 250 1300 80

g1 1090 300 180 700 10

g4 2500 2000 800 1600 30

wt 720 210 45 750 80

Values correspond to serum dilution required to yield z 50% reduction ofinfection by 8.104–105 RLU-equivalent promoting infection of pseudo-

typed viruses. Results correspond to the average of two experiments

performed in triplicate. The column #1 corresponds to the autologous serum

from patient #133. The columns #2–4 correspond to titrated HIV-1 human-

neutralizing sera. The column #5 corresponds to an HIV-1 negative serum.

infection was neutralized more efficiently (Table 2, columns

#1 to #4). These two mutants corresponded to the complete

deglycosylation of the V1-loop (g4) and to four mutations in

the blue cluster (g3B). The results displayed in the previous

section showed that these mutants were structurally and

functionally barely affected despite a large number of N-

glycans removed. For g1D (one mutation in D cluster) and

for g1 (two mutations but not in a cluster), a notable

neutralization effect for some sera was noticed. Only a

weak neutralizing activity was detected for HIV-1 negative

serum (10 < IT50 < 80; column #5).

Discussion

In the present report, we aimed at the determination of

the biological properties of Env glycomutants designed

from the available 3D structure. We used HIV-1 acute

infection primary isolates (PHI) to work with env sequences

from the early stage of the disease, as close as possible to

the transmission event. At this stage, viruses are still

homogenous in sequence (Delwart et al., 2002). Moreover,

the glycan shield could evolve during the course of infection

F. Reynard et al. / Virology 324 (2004) 90–102 97

by an escape process (Wei et al., 2003a) and it seemed

judicious to focus on primary sequences isolated early after

the infection. Alignment of 14 PHI gp120 allowed us to

select conserved glycosylation sites, which could be

grouped into four regions of occupancy when laid on a

3D model. We hypothesized that such a conservation and

clustering could reflect a role in shielding crucial gp120

epitopes during HIV-1 transmission. To check this hypoth-

esis, we engineered gp120 glycomutants with progressive

deglycosylation of each region. Each mutant was tested for

both its functional and antigenic properties and compared to

the wild-type protein.

The number of sequences analyzed was not sufficient to

be sure that the positions we chose as conserved are indeed

found in all HIV-1 isolates, but we can assume that these

positions are at least well conserved. Moreover, the ana-

lyzed sequences were almost exclusively from B clade

except two of them belonging to E and G clades (sequen-

ces #355AHT and #365AHT, respectively). Even if some

cohorts of acute seroconverters have been extensively

studied (Delwart et al., 2002; Freel et al., 2003; Hu et

al., 2000), they are few complete env sequences from PHI

available in HIV-1 sequences databases. A comparison

between our 14 gp120 PHI sequences and two other ones

found in databases (GeneBank accession nos. U32396 and

AF310108) showed that the N-glycan sites we selected

were actually present in these two PHI sequences (data not

shown). The repartition of the different conserved potential

glycosylation sites on the 3D model showed that N-glycans

could be located in specific regions. These regions are

virtual because of the arbitrary nature of our clusters: as the

available crystallized gp120 is partially deleted and its

association with the sCD4 and 17b Fab probably induced

conformational changes compared to gp120 alone, we

considered alternate possibilities to enlarge the definition

of each region. We chose to restrain the number of

deglycosylation to six because it had been previously

proposed that Env can tolerate around five mutations in

combination without disturbance of the infectivity (Ohgi-

moto et al., 1998; Reitter and Desrosiers, 1998).

Transient expression of the env glycomutants in the 293T

cells and subsequent Western blots analysis showed that

cleavage of the precursor gp160 into gp120 and gp41 was

affected by the number of N-glycans removed. Previous

studies have shown that removing of one N-glycan has no

impact on biosynthesis and functionality of gp160, but more

mutations (3–5) have major effects (Dirckx et al., 1990;

Fenouillet et al., 1994). In our hands, if one—or exception-

ally more mutations (g3B, g3C, and g4)—had generally

little impact on processing, the accumulation of three to six

mutations entailed a dramatic decrease in gp160 cleavage

and gp120/gp41 cohesion. This could be explained by the

misfolding of the partially deglycosylated protein or the

inefficient trafficking from endoplasmic reticulum to trans-

Golgi, where the cleavage by cellular endoproteases occurs

(Moulard and Decroly, 2000). These results are compatible

with previously published data (Johnson et al., 2001;

Quinones-Kochs et al., 2002; Reitter and Desrosiers,

1998). Both the number and the specific location of the

silenced N-glycans seemed to have additive effects. If some

N-glycans (especially at positions N109 and N148) play a

foreground role, others seem to be more accessory in terms

of functionality and antigenicity, notably those located on

the V1 variable loop and some in the B (e.g., N180/N185 or

N201/N208) or in the C cluster (N289, N253).

The positive results in cell-to-cell fusion assays indicated

that sufficient level of functional Env was expressed on the

cell surface. In this assay, fusion observed on GHOST-

CCR5 cells was inversely related to the number of N-

glycans removed. Previous studies have shown that all N-

glycans on V1 or V2 loops on HIV-1 primary isolate could

be mutated without affecting expression on the cell surface.

However, mutations reduced (four or five sites) or sup-

pressed (six sites) fusion activity (Quinones-Kochs et al.,

2002). The N-glycans on the V1/V2 stem and V3 loops have

often been described as playing an important role in HIV-1

strains coreceptor usage (Nakayama et al., 1998; Pollakis et

al., 2001). In our experimental conditions, no cell-to-cell

fusion was observed for the mutations N44 (V1/V2 stem)

and N148 (V3) when GHOST-CXCR4 cells were used as

indicators.

In order to characterize the effects of clustered N-glycans

mutagenesis on CD4 binding site, sCD4 binding was tested

by ELISA. It was barely affected by mutations. For some

mutants (g2C, g4C, and g5C), mutation at position N109

played an unfavorable role on sCD4 binding compared to

the results obtained on some constructs without mutation at

this position (g1C and g3C). Mutation N109 is located in

the C2 domain, 30 residues upstream the V3 loop. It has

been previously reported that N109Q substitution in HIV-1

is critical for the intracellular process of gp160, for the

ability of gp120 to bind to CD4, and for the infectivity by

reduction in the amount of gp120 incorporated in virions. It

has been proposed that such impairment could be due to

interactions disturbance between C2 and V3 regions (Willey

and Martin, 1993). Interestingly, the glycosylation site at

N109 is exceptionally well conserved in all Env primate

retroviruses (Kwong et al., 1998). Although g2C and g4C

were dramatically affected for sCD4 binding, they induced

syncytia formation in cell-to-cell fusion experiment on

GHOST CD4+–CCR5+. It could be tempting to conclude

that these mutants have acquired CD4 independence; how-

ever, cell-to-cell fusion assay on HOS CD4�–CCR5+ did

not show any syncytia.

Results obtained with pseudovirions indicated that the

incorporation efficiency depends on the number of glycans

removed. The correct processing of the precursor gp160

seems required for Env incorporation into viral particles

(Dubay et al., 1995; McCune et al., 1988). The uncleaved

form of the HIV-1LAI Env is highly presented at the cell

surface but particles following budding from the cell surface

are devoid of gp160 precursor (Moulard et al., 1999); other

F. Reynard et al. / Virology 324 (2004) 90–10298

data suggested that the entire gp120 with an adequate

conformation is required for Env complex incorporation

into virions (Li and Perez, 1997).

The mutants g3A and g1, despite cleavage alteration,

induced a high number of syncytia and promoted infec-

tious pseudoparticles formation. These results suggested

that the few gp160 that is correctly processed at the cell

membrane kept their functionality and could be incorpo-

rated into virions. The differences observed between fusion

assay and infectivity assay for mutants g2C and g4C could

be also imputed to the low density of functional Env found

on cell membranes, which has repercussions on virion

envelope composition. Detection of gp41, but not gp120,

on pseudovirions g2C and g4C provided the confirmation

of N109 influence upon Env edifice stability; the not yet

shedded gp120/gp41 edifices present at the cell surface

(which induced fusion in cell-to-cell fusion assay) would

be incorporated into pseudovirions, but once in pseudovi-

rions, the gp120 would shed. The cell-to-cell fusion assay

is a very sensitive test for monitoring Env functionality

because in theory, a single functional Env oligomer must

be sufficient to promote membranes fusion on susceptible

cells and therefore to lead to the formation of a detectable

fusion event. This could explain why some mutants

showed cell fusion although the gp120 could not be

detected by immunoblot.

With one unique mutation at position N44, the mutant

g1Awas correctly processed. Unexpectedly, a weak level of

cell-to-cell fusion and infection was observed. Previous

studies suggested that the glycan located at N44 influences

the spacial location of the V1/V2 loop of HIV-1ADAglycoprotein and consequently modulates the accessibility

of the coreceptor binding site (Kolchinsky et al., 2001). We

propose that for g1A, the mutation relocates V1/V2 the

same way but resulting in a reduced accessibility of the

CCR5 binding site.

Early studies showed that the acquisition or the removal

of glycans in variable loops of HIV-1 or SIV modify the

sensitivity of these viruses to neutralization (Bolmstedt et

al., 1996; Chackerian et al., 1997; Matsushita et al., 1988).

In our hands, the most remarkable results were obtained on

mutants devoid of three N-glycans in V1 or 4 N-glycans in

C3 (g4 and g3B, respectively). It has been suggested that the

density of the envelope spikes on the surface of virions can

influence relative sensitivity to antibody-mediated neutrali-

zation. If mutations result in a decrease in spikes incorpo-

ration or in a high level of spontaneous shedding, then the

increased neutralization sensitivity could reflect a reduction

in the number of antibodies required to inhibit mutant

virions (Burton et al., 2000). However, immunoblots made

on pseudotyped particles showed that neutralization sensi-

tivity was independent on the intensity of the gp120 signal.

For V1 deglycosylation (g4, three mutations), there is

apparently a few differences comparing wt Env. However,

g4 pseudotyped particles are more sensitive to human-

neutralizing sera. The V1/V2 loop is a bulky domain that

emerges from the gp120 core; moreover, V1 loop is struc-

tured by three disulfide bridges: this particular topology

could explain why deglycosylation led to small differences

in structure and processing comparing parental glycopro-

tein. However, a complete deglycosylation of the V2 loop

(g5, 2 mutations) caused a drastic impairment for gp160

cleavage, conformation, and CD4 binding. Both structures

have complex-type sugar linked to asparagine (and an extra

glycan not yet characterized, for V1). Apparently, the

chemical nature of glycans does not explain differences

observed between the two mutants.

Thus, we can hypothesize that some sugars play an

essential role during Env biosynthesis and processing

(e.g., sugars linked at residues N109 and N148); the

silencing of these sugars entails defects on cleavage or

gp120/gp41 stability. A second type of sugar plays an

accessory role in envelope processing but probably serves

to shield protein against antibodies by evolving during the

course of the disease. The sugars removed from g3C, g4,

and g3B mutants would belong to this second type. This

hypothesis agrees with a previous study showing that N-

glycans important for infectivity were not randomly distrib-

uted on HIV-1HXB2 gp120, and consequently, the role of the

remaining N-glycans would be to evade the host immune

response (Lee et al., 1992). Interestingly, N-glycan sites

silenced on g4 and g3B are separated by few residues (less

than eight amino acids) and could be considered as redun-

dant. Moreover, the asparagine residues mutated for g3C or

g3B locate close together on 3D model. This special

topology strengthens the hypothesis of evolving and adapt-

able positions for N-glycans shield in face of the antibody

repertoire.

Recent results suggested that high-mannose residues

defining 2G12 epitope play a foreground role in DC-SIGN

or related lectins binding (Sanders et al., 2002). The 2G12

human monoclonal antibody neutralizes HIV-1 in vitro and

protects macaque in association with others antibodies in

passive immunization experiment from Simian-HIV-1

(SHIV) virus challenge (Mascola et al., 2000; Trkola et

al., 1996). The N-glycans N180–N185 from the B region

take part in 2G12 epitope. They are silenced in g3B mutant.

Thus, we can also hypothesize that in this area, the presence

of a patch of N-glycans has a critical importance on virus–

cell interactions. Consequently, antibodies directed against

this region of Env could interfere with lectins binding and

thus could allow better viruses neutralization.

The differences between the mutants and wt protein are

not the result of gross defect in expression or processing of

the envelope complex because the majority of mutants were

cleaved and exported to the cell surface. The cleavage and

the conformation disturbance are apparently independent of

clustering mutations or glycan type. However, results can be

modulated in function of the respective position of each

glycan removed on the gp120, underlining the different role

of each sugar in the Env protein characteristics. Although

our integrated approach, taking into account the 3D disper-

F. Reynard et al. / Virology 324 (2004) 90–102 99

sion of N-glycans on gp120 from PHI, failed to clearly

demonstrate a cluster-specific role of N-glycans in Env

function and immunogenicity, it has led us to identify new

potential immunogens. If the criteria in the choice of a Env

immunogen suitable for neutralizing antibodies induction

are based on the preservation of the structure as much as

possible, but with an enhanced exposition of critical epito-

pes and correlatively enhanced neutralization, the g3B and

g4 mutants are good candidates.

Materials and methods

Sequence data

Nucleotide sequences coding for the entire gp160 env

region were generated from DNA PBMC from patients

with acute HIV-1 infection. These sequences were de-

scribed in a previous study (Ataman-Onal et al., 1999).

They were named 133 AHM, 146 AHM, 153 AHM, 159

AHM, 160 AHM, 374 AHM, 384 AHM, 385 AHM

(homosexual transmission), 120 AHT, 309 AHT, 355

AHT, 365 AHT, 373 AHT, and 426 AHT (heterosexual

transmission). All sequences were aligned with CLUS-

TALW (MacVector software, Oxford Molecular Group).

The 3D model was based on the coordinates for the HIV-

CD4-17bFab antibody complex deposited in the Protein

Data Bank as entry IG9M. Molecules were drawn with

PDBwiever (Glaxo-Smithkline).

Site-directed mutagenesis

Wild-type 133 env sequence was cloned into the Xho1–

Xba1 sites of the pCI expression vector (Promega, Madison,

USA) using standard protocols. To remove potential N-

glycosylation sites, asparagine to glutamine substitutions

were carried out using the QuickChange site-directed mu-

tagenesis kit (Stratagene, La Jolla, USA). All mutant con-

structs were checked by sequencing both strands on a 377

automatic sequencer (Perkin-ABI, Boston, USA) with the

PRISM Big Dye terminator V.02 (Perkin-ABI). Plasmid

DNA was prepared using the NucleobondPC500 kit

(Macherey-Nagel, Düren, Germany).

Cells transfection and pseudovirions generation

293T cells were used for transfection experiments and

were cultivated in MEM (Gibco-LifeTech, Gaithersburg,

USA) supplemented with 10% fetal calf serum, 1% nones-

sential amino acids and 1% Na-pyruvate (Gibco-LifeTech).

Cells (8.105) were cotransfected with 0.2 Ag pCI-rev and 1Ag of the different pCI-env constructs using the Lipofect-AMINE Kit (BRL, Rockville, USA). The env gene cloned

in reverse orientation in the same vector (pCI-anti) was used

as a negative control. Cells were harvested 24 h (for cell-to-

cell fusion assay) or 48 h later (for both immunoblots and

ELISA). Alternatively, pseudovirions stocks with wild-type

or env mutants were generated as previously described with

the help of the env-deficient viral reporter vector pNL43-

LucR-E- (Park et al., 1998). Supernatants containing pseu-

doviruses were harvested 48 h post-transfection and frozen

at �80 jC. The amount of virus particles was determined byVidas-p24 tests (bioMerieux, Marcy-l’Etoile, France).

In vitro cell-to-cell fusion assay and infectivity assay

Twenty-four hours post-transfection, 293T cells were

harvested and replated at 105 cells in a 12-well plate. The

unseeded cells were used to check gp120 expression by an

immunoblot. Cells were left to re-adhere during 6 h, then

GHOST-CD4+CCR5+,GHOST-CD4+CXCR4+, or HOS-

CD4�CCR5+ cells were added (106 cells/well) and incubat-

ed during 16 h. The cells were fixed 15 min in 0.5%

glutaraldehyde, colored in May–Grünwald/Giemsa stain,

and syncytia were observed with an optical microscope.

Alternatively, 106 GHOST cells were infected by pseudovi-

ruses (50 ng p24/wells). After 12 h of incubation, inoculum

was replaced by 1 ml of fresh culture media. After 72 h, the

cells were lysed and luciferase activity was measured with

the Luciferase Assay Kit (Promega) on a luminometer (TD

20/20, Turner).

Western blot

The same protocol was used for both cell lysates, culture

supernatants, and pseudoparticles pellets. Forty-height hours

post-transfection, cells were harvested, washed once with

PBS1x, lysed in TBS1x/NP40 1%, and finally cleared by

centrifugation (15000 g/10 min). The culture supernatants

were cleared of the cell debris by centrifugation (15000 g/10

min). The pseudoparticles (equivalent to 500 ng of p24) were

pelleted 3 h at 20 000 g on a 20% sucrose cushion and then

lysed by 50 Al of TBS1x/NP40 1%. The proteins were heat-denatured in presence of h-mercaptoethanol and separatedon a 8% SDS-polyacrylamide gel. After electro-transfer, the

membrane (Hybon-P; Amersham-Pharmacia, UK) was sat-

urated in PBS1x/Tween 200.1% nonfat dry milk 3% and

incubated overnight at 4 jC with a goat anti-gp120 poly-clonal antibody (Biogenesis ref. 5000–0557, Poole, UK; 1

Ag/ml). Alternatively, an anti-gp41 monoclonal antibody(F240, NIH AIDS Research and Reference Reagent Pro-

gram, Rockville, USA) was used at 1 Ag/ml. After extensivewashing, the membrane was incubated for 45 min at room

temperature with peroxydase-conjugated antibodies (Jack-

son, West Grove, USA; 0.1 Ag/ml). Detection was performedusing the ECL reagent (Amersham-Pharmacia; UK). Sam-

ples were normalized by densitometry on a Typhoon 8600

reader (Amersham-Pharmacia, UK) based on the detection of

the Rev and Actin proteins on immunoblots with monoclonal

antibodies against Rev (1F6D1B7; bioMérieux, Marcy-

L’Etoile, France) and against human actin (A4700; Sigma-

Aldrich, St. Louis, USA).

rology 324 (2004) 90–102

ELISA

Env ELISA-capture was carried out on cell-culture

supernatants and cell lysates processed as described above.

The D7324 polyclonal antibody (Aalto, Dublin, Ireland)

was used as capture antibody as previously described

(Brand et al., 2000). After normalization of total protein

concentration with the BCA protein assay kit (Pierce,

Rockford, USA), cell lysates were diluted at 1:10 in

TBS/NP40 1%/New-Born Calf Serum 2%. For detection,

the autologous HIV + serum from #133 patient (B clade;

hôpital de la Croix-Rousse; Lyon, France) was used at 1/

100 dilution. Alternatively, soluble CD4 (sCD4, NIH AIDS

Research and Reference Reagent Program) was used at a

final concentration of 0.5 Ag/ml. The soluble CD4 wasdetected with the BAF 379 monoclonal antibody (R&D,

Abington, UK) directed against its V4 loop. Each result

corresponded to the mean of two independent experiments

made in duplicate.

Neutralization assay

Supernatant containing pseudotyped particles were pre-

incubated for 1 h at 37 jC with four serial human-neutralizing sera or one nonneutralizing human serum

(Burrer et al., 2001) before infection of GHOST-CCR5

cells in triplicate wells in 96-well plates. The volume of

supernatant loaded in each well corresponded to the quan-

tity of pseudotyped particles necessary to promote 80000–

100000 relative light units (RLU) after infection of per-

missive cells. After an overnight incubation, the medium

was replaced and incubation took place during additional 2

days. After cell lysis, the infectivity was measured by using

LucLite Plus kit (Perkin–Elmer) and luminescence was

read on a TopCount apparatus (Packard Biosciences).

Neutralizing titers were expressed as the reciprocal of the

plasma dilution required to protect 50% of cells from

pseudoviruses infection.

F. Reynard et al. / Vi100

Acknowledgments

We thank Dr. Christophe Guillon and Dr. Yasemine

Ataman-Önal for helpful discussion and manuscript reading.

The sCD4 and the monoclonal antibody F240 (Dr. Posner)

were obtained through the NIH AIDS Research and

Reference Reagent Program. We thank C. Moog (INSERM

U544, Strasbourg) for neutralizing and control sera. This

work was supported by ANRS (#2003/006) and bioMérieux

SA.

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HIV-1 acute infection env glycomutants designed from 3D model: effects on processing, antigenicity, and neutralization sensitivityIntroductionResultsRational of deglycosylation constructsInfluence of N-glycans removal on gp160 cleavage and gp120 sheddingInfluence of N-glycans removal on cell membrane presentation and incorporation in pseudovirionInfluence of N-glycan removal on sCD4 bindingNeutralizing activity against pseudoparticles bearing Env glycomutants

DiscussionMaterials and methodsSequence dataSite-directed mutagenesisCells transfection and pseudovirions generationIn vitro cell-to-cell fusion assay and infectivity assayWestern blotELISANeutralization assay

AcknowledgementsReferences