Conserved Role of an N-Linked Glycan on the Surface Antigen of Human Immunodeficiency Virus Type 1 Modulating Virus Sensitivity to Broadly Neutralizing Antibodies against the Receptor and Coreceptor Binding Sites.

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Product# 1039 HIV-1 YU2 (M Tropic)Envelope Glycoprotein gp41

Product# 1081 HIV-1 gp120 (ADA)

Product# 1011 HIV-1 gp120 (subtype C)

Product# 1031 HIV-1 gp120 (YU2)

ABSTRACT

HIV-1 establishes persistent infection in part due to its ability to evade host immune responses. Occlusion by glycans contributes to masking conserved sites that are targets for some broadly neutralizing antibodies (bNAbs). Previous work has shown that removal of a highly conserved potential N-linked glycan (PNLG) site at amino acid residue 197 (N7) on the surface antigen gp120 of HIV-1 increases neutralization sensitivity of the mutant virus to CD4 binding site (CD4bs)-directed antibodies compared to its wild-type (WT) counterpart. However, it is not clear if the role of the N7 glycan is conserved among diverse HIV-1 isolates and if other glycans in the conserved regions of HIV-1 Env display similar functions. In this work, we examined the role of PNLGs in the conserved region of HIV-1 Env, particularly the role of the N7 glycan in a panel of HIV-1 strains representing different clades, tissue origins, coreceptor usages, and neutralization sensitivities. We demonstrate that the absence of the N7 glycan increases the sensitivity of diverse HIV-1 isolates to CD4bs- and V3 loop-directed antibodies, indicating that the N7 glycan plays a conserved role masking these conserved epitopes. However, the effect of the N7 glycan on virus sensitivity to neutralizing antibodies directed against the V2 loop epitope is isolate dependent. These findings indicate that the N7 glycan plays an important and conserved role modulating the structure, stability, or accessibility of bNAb epitopes in the CD4bs and coreceptor binding region, thus representing a potential target for the design of immunogens and therapeutics.

IMPORTANCE N-linked glycans on the HIV-1 envelope protein have been postulated to contribute to viral escape from host immune responses. However, the role of specific glycans in the conserved regions of HIV-1 Env in modulating epitope recognition by broadly neutralizing antibodies has not been well defined. We show here that a single N-linked glycan plays a unique and conserved role among conserved glycans on HIV-1 gp120 in modulating the exposure or the stability of the receptor and coreceptor binding site without affecting the integrity of the Env in mediating viral infection or the ability of the mutant gp120 to bind to CD4. The observation that the antigenicity of the receptor and coreceptor binding sites can be modulated by a single glycan indicates that select glycan modification offers a potential strategy for the design of HIV-1 vaccine candidates.

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INTRODUCTION

Although the role of neutralizing antibodies has yet to be determined in the only clinical trial of human immunodeficiency virus type 1 (HIV-1) vaccines that has shown a modest degree of protection, it is generally believed that it would be advantageous for a vaccine to elicit broadly neutralizing antibodies (bNAbs) against diverse primary isolates. Passive administration of neutralizing monoclonal antibodies (MAbs) or bNAbs derived from HIV-1-infected patients has been shown to protect macaques from simian-human immunodeficiency virus (SHIV) infection (1,–5). A fraction of HIV-1-infected individuals (∼20 to 30%) generate bNAbs within 2 to 4 years of initial infection (6,–10). However, generation of bNAbs by active immunization has been a challenge, as no HIV-1 vaccine candidate has successfully elicited antibodies with a similar neutralizing breadth (8, 11).

Nevertheless, broadly neutralizing monoclonal antibodies isolated from selected individuals have helped define the targets of bNAbs. These bNAbs are directed against one of five conserved epitopes on HIV-1 envelope glycoprotein (Env); the CD4 binding site (CD4bs), the membrane-proximal ectodomain region (MPER), carbohydrates on the outer domain, a quaternary structure in the V1 and V2 loops, and a newly described epitope present only in cleaved envelope trimers (7, 11,–13). However, HIV-1 has evolved many protective mechanisms to evade host immune responses, including occlusion of potential targets by glycans (14,–20). These glycans account for ∼50% of the molecular mass of the Env surface antigen (gp120) and may mask nearly the entire surface of Env, including the conserved epitopes targeted by some bNAbs, rendering it difficult to elicit antibodies targeting these sites.

We and others previously reported that removal of a single N-linked glycan located at amino acid position N197 (N7) on gp120 resulted in significantly increased sensitivity of HIV-1 to neutralization (21,–23) and enhanced the ability of Env to induce neutralizing antibodies in macaques (21). However, these studies were based on a small number of isolates. Since the N7 glycan is highly conserved across diverse HIV-1 and simian immunodeficiency virus (SIV) isolates (19, 23, 24), it is of interest to determine if the N7 glycan plays a conserved role in the antigenicity, immunogenicity, and function of HIV-1 Env.

In the present study, we sought to extend earlier observations by examining if the role of the N7 glycan is unique among potential N-linked glycans (PNLGs) in the conserved region of gp120 and determining if the effect of the N7 glycan on Env antigenicity is conserved among diverse isolates of HIV-1. Specifically, we used broadly neutralizing monoclonal antibodies with defined epitope specificities to study the effects of the N7 glycan on the antigenicity of Env from a panel of HIV-1 strains representing diverse clades, tissue origins, coreceptor usages, and neutralization sensitivities. Results from this study indicate that the N7 glycan has a unique and conserved role modulating the exposure or stability of conserved epitopes in the CD4bs and variable loops.

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MATERIALS AND METHODS

 

Construction of N-linked glycosylation site mutants.

Plasmids containing the rev-env genes of individual HIV-1 isolates were used as the template for site-directed mutagenesis using a QuikChange mutagenesis kit (Stratagene). Plasmid encoding QA255.662J Env were kindly gifted by Julie Overbaugh (25). Plasmids encoding 89.6 and ADA were gifted by Joseph Sodroski (26, 27). Plasmids encoding B33, LN40 (28), and JR-FL (29) Envs were gifted by Paul Clapham. A plasmid encoding SF162 was gifted by Leonidas Stamatatos and Cecilia Cheng-Mayer (30), and 1084i was gifted by Ruth Ruprecht (31). PVO.4 was obtained through the NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH, from David Montefiori and Feng Gao (32).

To generate glycan mutants of Env, the asparagine (N) residue in the PNLG sequon (N-X-S/T) encoded by most env genes was substituted with glutamine (Q) by site-specific mutagenesis. B33 N197 was replaced with glutamic acid (E) because the N197Q mutant Env did not generate viable virus. The N197 PNLG sequon was restored in QA255 by replacing the proline (P) at position 199 with serine (S) and in JR-FL Env by replacing the aspartic acid (D) at position 197 with N. Conditions for the mutagenesis included a two-step PCR: 95°C for 2 min followed by 30 cycles of annealing and extension at 55°C for 30 s and denaturing at 95°C for 20 s and, finally, annealing at 65°C for 6 min. The mutated rev-env genes were then subcloned into mammalian expression vector pSVIIIΔEnv (33). The results of site-directed mutagenesis were verified by DNA sequencing.

 

Monoclonal antibodies and polyclonal serum.

The following monoclonal antibodies were obtained from the NIH AIDS Research and Reference Reagent Program: MPER-targeted antibody 4E10 (34) and mannan-targeted antibody 2G12 (35, 36) were from Hermann Katinger, CD4bs-targeted antibody IgG1b12 was from Dennis Burton and Carlos Barbas (37,–40), V3 loop-targeted antibody 447-52D (41,–46) and V2 loop-targeted antibody 697-30D (46,–48) were from Susan Zolla-Pazner, and CD4-induced antibody 17b (49,–54) was from James E. Robinson. Recombinant soluble CD4-183 (sCD4-183), obtained from Pharmacia, Inc., contains the first two domains of human CD4 produced in Escherichia coli (55). CD4bs-targeted antibody VRC01 (56) was obtained from John Mascola, and V1 and V2 loop-targeted antibodies PG9 and PG16 (57) were from Dennis Burton. The CD4-IgG2 was from Progenics Pharmaceuticals, and C5-targeted antibody D7324 was purchased from Aalto BioReagents. The pooled clade B serum was from NABI, and the pooled clade C serum was a gift from Charles Wood.

 

Infectivity and neutralization assays.

Stocks of Env-pseudotyped viruses were prepared by cotransfection of Env and backbone plasmid in 293T cells (32). pSG3ΔEnv backbone was used to generate clade B Env-pseudotyped virus, and Q23ΔEnv backbone was used to generate clade A and C Env-pseudotyped viruses. All assay stocks were titrated in TZM-bl cells as previously described (32). Infectivity of Env-pseudotyped virus was measured by luciferase reporter gene expression in TZM-bl cells. Neutralization as measured by reduction of luciferase gene expression was performed as previously described (22, 58). Briefly, indicator virus containing 150 50% tissue culture infective doses (TCID50) was incubated with serial 3-fold dilutions of antibody or serum samples in duplicates in a total volume of 150 μl for 1.5 h at 37°C in 96-well flat-bottom culture plates before being added to TZM-bl cells (10,000 cells in 100 μl of growth medium containing DEAE-dextran). One set of control wells received cells plus virus (virus control), and another set received cells only (background control). After a 72-h incubation, 100 μl of cells was transferred to a 96-well white solid plate (Costar) for assays of luciferase activities using BrightGlo substrate solution as described by the supplier (Promega). Neutralization activity was expressed as the concentration of antibodies or reciprocal serum dilution that resulted in 50% reduction (IC50) of relative light units (RLU).

 

Fortebio Octet biolayer interferometry.

Fortebio Octet biolayer interferometry was performed as previously described (59). Briefly, anti-human IgG Fc capture tips (Fortebio) were prewet for 5 min in HBS-EP running buffer (GE Healthcare) immediately prior to use. The microplates used in the Octet were filled with 200 μl of sample or buffer per well and agitated at 1,000 rpm. Tips were then saturated with 10 μg/ml of CD4-IgG2 and then washed with running buffer before being transferred into 2-fold dilution series of gp120, where the association rate constant (ka) (1/Ms) was read. The tips were then transferred into running buffer to measure the dissociation rate constant (kd) (1/s). The tips were regenerated using 0.1 M glycine, pH 1.5 (GE Healthcare), before being used on subsequent samples. Values from a CD4-IgG2-coated tip that was transferred into running buffer alone were subtracted from all test values. Additionally, one uncoated tip was incubated with gp120 alone to ensure that there were no interactions between Env and the capture tips. All interaction analyses were conducted at 25°C.

 

Viral protein capture assays.

The p24 concentration of a given pseudovirus preparation was measured with an HIV-1 p24 enzyme-linked immunosorbent assay (ELISA; Zeptometrix) and compared to its TCID50. Env capture assays using D7324 were carried out as previously described (43, 60), with slight modifications. In brief, 96-well plates were coated with 10 μg/ml of sheep anti-gp120 capture antibody D7324 (Aalto Bio Reagents Ltd., Dublin, Ireland) overnight at room temperature, blocked with phosphate-buffered saline (PBS)–0.05% Tween–5% milk, washed with PBS–0.05% Tween, and incubated for 2 h at 37°C with wild-type (WT) or N7 mutant Env from one of three sources: gp120 generated by recombinant vaccinia virus-infected cells (61), detergent-lysed pseudovirus, or whole pseudovirus. SF162 or JR-FL Env captured on plates was washed before monoclonal antibody (MAb) 697-30D (at 30 μg/ml or 3 μg/ml, respectively) was added and incubated for 1.5 h at 37°C. After a washing, the bound MAbs were detected using horseradish peroxidase (HRP)-conjugated anti-human IgG (Thermo Fisher) followed by incubation with TMB substrate (Sigma). After 2 M H2SO4 was used to stop the reaction, MAb binding was quantified by optical density readings at 450 nm (OD450). Binding of antibody to each virus was normalized to OD450 readings obtained with HIV-positive polyclonal serum for the same virus. This controlled for possible differences in the number of virions captured. Separate wells included supernatant from parental vaccinia virus-infected cells or ΔV2 gp120 as negative controls (data not shown). Binding of WT virus was included on each plate and set as 100% binding. All experiments were repeated three times in duplicates.

 

Statistical analysis.

Prism (GraphPad Software, Inc.) was employed for all statistical analyses, all using two-sided tests. The Wilcoxon matched-pairs signed-rank test was used to compare equilibrium dissociation constant (KD), picograms of p24/TCID50, and percent binding values.

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RESULTS

 

The N7 glycan is unique among PNLGs in the conserved regions of gp120 in its ability to modulate the sensitivity of HIV-1 to CD4bs-specific antibodies.

Previous work demonstrated that the N7 glycan modulates sensitivity of HIV-1 to CD4bs-specific neutralizing antibodies (21,–23). To examine if the same effect is exerted by other conserved glycans, we introduced mutations replacing the asparagine (N) residue with glutamine (Q) at each of the PNLG sequons (N-X-T/S) in the conserved regions of surface antigen gp120 of HIV-1 89.6. For this study, we focused on mutant Env proteins that are expressed and processed normally and retain overall functional integrity as indicated by successful rescue of viable virus (data not shown). The N262 glycan mutant did not generate viable virus and thus was not included in further experiments. Neutralization sensitivity of pseudotyped viruses bearing WT or mutant Env was examined in a TZM-bl cell assay against sCD4, CD4bs, and MPER-directed antibodies as well as pooled HIV-positive serum. Results in Fig. 1 show that the N7 glycan is unique among PNLGs in the conserved regions of gp120 in its ability to modulate virus sensitivity to CD4bs-specific neutralizing antibodies. Removal of selected single N-linked glycans modified the sensitivity of virus to certain antibodies; e.g., the N361 mutant was modestly more sensitive to IgG1b12. However, only removal of the N7 glycan increased the sensitivity of 89.6 to all CD4bs-directed antibodies tested as well as HIV-positive pooled serum (21). This indicates that the N7 glycan plays a unique role among conserved glycans on gp120, modulating the stability of or access to the CD4bs in the 89.6 Env

FIG 1

Neutralization sensitivity of viruses pseudotyped with WT 89.6 Env or PNLG mutants in the conserved regions of Env. Mutants were designated by the position of the asparagine residue of the PNLG sequon. The N197 mutant is abbreviated as N7. Neutralization assay was performed using sCD4, CD4-IgG, broadly neutralizing monoclonal antibodies, or pooled HIV-positive serum at concentrations or serum dilutions as indicated. Neutralization was quantified as the percent inhibition of viral infectivity measured by RLU expressed in TZM-bl indicator cells. The dotted line shows the concentration or reciprocal serum dilutions that resulted in 50% inhibition of viral infectivity (IC50).

 

Increased sensitivity of N7 mutant viruses to CD4bs-specific neutralizing antibodies is conserved among diverse isolates of HIV-1.

Previous work examined the role of the N7 glycan in only a limited number of isolates (21,–23). Because the sequon for the N7 glycan is present in more than 95% of available HIV and SIV sequences (19, 23, 24), we examined if the N7 glycan plays a conserved role in diverse isolates of HIV-1. To test this, we modified the N7 glycan site on the Env from a panel of HIV-1 strains representing different clades, tissue origins, coreceptor usages, and neutralization sensitivities (Table 1), including two viruses, QA255 and JR-FL (25, 29), that lack the N7 glycan site in the Env of WT viruses. To determine the effects of the N7 glycan on the panel of Env, we used site-directed mutagenesis to remove the N7 glycan site of most Env proteins by replacing the asparagine (N) with a glutamine (Q), or to restore the N7 glycan site for those Env missing this site in their WT sequences (see Materials and Methods). Plasmids expressing WT or N7-modified Envs were then used to generate pseudoviruses. The neutralization sensitivity of these pseudoviruses was first tested using antibodies that recognize CD4bs or CD4 induced (CD4i) epitopes (Table 2).

TABLE 1

Selected HIV-1 isolates representing different clades, coreceptor usages, and tissue origins

Isolate

Clade

Tropism

Source

Reference

Accession no.a

QA255

A

R5

PBMCb

25

NA

89.6

B

R5/X4

PBMC

101

U39362

ADA

B

R5

PBMC

100

AY426119

B33

B

R5

Brain

28

ADD17640

LN40

B

R5

Lymph node

28

ADD17641

JR-FL

B

R5

Brain

29

AAB05604

PVO.4

B

R5

PBMC

32

AAW64259

SF162

B

R5

Brain

99

P19550

1084i

C

R5

PBMC

31

AAV80387

aNA, not available. Accession numbers are from GenBank.

bPBMC, peripheral blood mononuclear cells.

TABLE 2

Neutralization sensitivity of WT and N7 mutant pseudotyped viruses to monoclonal antibodies targeting CD4bs or CD4-induced (CD4i) epitopes

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FIG 2

V2 loop amino acid sequence of the panel of HIV-1 isolates. The amino acid sequence of the V2 loop for each Env (252829313299,–101) was aligned to the HXB2 reference sequence (102) using ClustalW. Numbering is based on the HXB2 amino acid sequence. Identical amino acid residues are indicated by dots. Dashes indicate gaps. PNLG sites in each isolate are indicated by a red letter N. The N7 PNLG site is at amino acid position 197.

It has been reported that glycan modification in conserved regions of HIV-1 gp120 reduced viral infectivity (212363). To determine if the increased sensitivity of N7 mutants to CD4bs antibodies is associated with reduced infectivity of the mutant viruses, we examined the infectivity of the N7 glycan mutant viruses that showed the greatest increase in neutralization sensitivity to CD4bs antibodies compared to their WT versions. We measured the TCID50 of these pseudotyped viruses normalized to their p24 content. Results in Fig. 3 show that the removal of the N7 glycan did not impact the infectivity of these viruses, supporting the notion that the increased neutralization sensitivity of N7 glycan mutants to CD4bs antibodies was not due to their decreased infectivity

.
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FIG 3

Infectivities of viruses pseudotyped with WT or mutant N7 Env. The relative infectivities of viruses pseudotyped with WT or N7 mutant Env from ADA, B33, or PVO.4 were measured as the quantity of p24 (in picograms) normalized to the infectivity (TCID50) of the respective viruses. Solid bars indicate viruses pseudotyped with the N7-glycosylated form of Env and hatched bars the N7-deglycosylated forms. Results for all viruses are shown, with the average and standard deviation obtained from three independent experiments performed in duplicates.

 

Removal of the N7 glycan does not alter the kinetics of binding of gp120 to CD4.

To determine if N7 glycan removal alters the affinity of Env protein to its receptor, we examined the kinetics of binding of CD4-IgG2 to gp120 proteins with or without the N7 glycan. Monomeric gp120 proteins from ADA, JR-FL, and PVO.4 were purified as previously described (61), and their kinetics of binding to CD4-IgG2 were compared by biolayer interferometry (Table 3). Different gp120 proteins showed different KD values, in agreement with observations from previous studies (64,–66). However, the presence or absence of the N7 glycan did not affect the KD values of gp120, indicating that N7 glycan mutations did not alter the properties of binding of monomeric gp120 to its receptor, consistent with the lack of effect of the N7 glycan on viral infectivity.

TABLE 3

Binding affinity of N7-glycosylated and deglycosylated gp120 to CD4-IgG2

Env

N7 glycana

KD (M)

ka (1/Ms)

kd (1/s)

P valueb

ADA

+

4.74E−09

5.13E + 04

2.46E−04

0.25

6.85E−09

4.21E + 04

2.91E−04

JR−FL

+

3.64E−09

7.19E + 04

2.61E−04

0.25

2.72E−09

6.69E + 04

1.81E−04

PVO.4

+

4.02E−08

4.59E + 04

1.80E−03

>0.99

4.01E−08

5.52E + 04

2.20E−03

aN7-glycosylated (+) and deglycosylated (−) gp120s were used.

bP value for KD (equilibrium dissociation constant) (M) obtained with N7-glycosylated or deglycosylated gp120 is calculated by the Wilcoxon matched-pairs signed-rank test.

 

Effect of the N7 glycan on virus sensitivity to CD4-induced neutralizing antibodies.

It has been demonstrated that removing the N7 glycan increases the sensitivity of HIV-1 not only to CD4bs-directed antibodies but also to CD4-induced (CD4i) neutralizing antibodies, potentially by enhancing the stability of or access to epitopes recognized by CD4i antibodies (22). Furthermore, CD4-independent viruses, which presumably have Env in an open conformation upon CD4 binding, are significantly more sensitive to neutralization by sCD4, similar to our finding with N7-deglycosylated viruses (21, 22). Therefore, we examined the sensitivity of the panel of HIV-1 strains to CD4i antibody 17b in the presence or absence of subneutralizing concentrations of sCD4 (Table 2). While the N7 glycan modulated sensitivity of virus to CD4bs antibodies, its effect on viral sensitivity to CD4i antibodies appeared to be isolate dependent. Increased neutralization sensitivity to 17b was observed only in N7 mutants of 89.6, ADA, and LN40 N197S. The N7 mutant of a tier 3 isolate, PVO.4 (67), was only neutralized by 17b, with IC50s close to 20 μg/ml, which was the upper limit of the assay, making it difficult to determine if the N7 glycan has any effect on neutralization sensitivity to 17b. All other blood and lymph node viruses tested, either with or without the N7 glycan, were resistant to neutralization by 17b, in either the presence or absence of sCD4.

For the few CNS isolates tested, N7 glycan removal did not modulate the neutralization sensitivity to 17b; however, we also observed an isolate-dependent effect of the N7 glycan on virus sensitivity to neutralization by 17b. The presence of subneutralizing concentrations of sCD4 augmented the neutralizing sensitivity of B33 to 17b, but no change was seen in SF162 or JR-FL. JR-FL was resistant to neutralization by the highest concentration of 17b used in the presence or absence of sCD4.

We also investigated if the increased sensitivity to CD4i antibodies correlated with the ability of the mutant viruses to infect CD4-negative cells. Infectivity of pseudotyped viruses bearing WT or mutant Env on CD4-negative cells (NNP2-CCR5 for R5-tropic viruses or U87-CXCR4 for 89.6 [21]) was determined using inocula normalized for their infectivity in isogenic CD4-expressing cells (NP2-CCR5-CD4 or U87-CXCR4-CD4, respectively) (Fig. 4). Increased infectivity in CD4-negative cells was observed only in ADA N197S and 89.6 N7 glycan mutants. Therefore, the ability of N7 mutant Env to mediate CD4-independent viral entry appears to be isolate specific and dependent on the specific amino acid substitutions at the PNLG site.

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FIG 4

CD4 dependence of infection by WT and N7 glycan mutants. The infectivities of WT and N7 mutants were compared in cells expressing coreceptor CCR5 or CXCR4, with or without coexpression of CD4. The infectivity of R5 tropic viruses was determined in NP2 cells expressing CCR5. The infectivity of 89.6 was determined using U87 cells expressing CXCR4 (adapted from the method of Li et al. [21]). The inoculum used for each virus was normalized by its infectivity in isogenic cells expressing the CD4 receptor (200,000 RLU, measured in NP2-CCR5-CD4 or U87-CXCR4-CD4 cells). Solid bars indicate infectivity of viruses with the N7 PNLG present, hatched bars indicate N197Q mutants, and the striped bar indicates ADA N197S mutant virus. The results in all panels represent the averages from at least two independent experiments performed in duplicates.

 

Effect of the N7 glycan on neutralization sensitivity to V3-specific monoclonal antibodies.

The increased susceptibility of the N7 mutants to CD4i antibodies indicates the possibility of enhanced exposure or stability of the bridging sheet. Indeed, previous work has shown that removing the N7 glycan results in increased sensitivity of HIV-1 to antibodies targeting the V3 loop and MPER (21, 22). Additionally, the crystal structure of soluble, cleaved trimers indicates that the crown of the V3 loop is buried under the N7 glycan of an adjacent protomer (68, 69). We examined if the removal of the N7 glycan correlated with increased exposure or stability of neutralizing epitopes in the V3 loop, which is a major determinant of coreceptor usage. Neutralization sensitivity to 447-52D, an antibody that recognizes the tip of the V3 loop (42), was determined for pseudotyped viruses with or without the N7 glycan (Table 4). Interestingly, removal of the N7 glycan increased the susceptibility of all viruses neutralized by 447-52D, including the CNS-derived isolates. Even PVO.4 and JR-FL, two isolates that are resistant to 50 μg/ml of 447-52D when the N7 glycan is present, were readily neutralized by the same antibody when the N7 glycan was removed. This indicates that the N7 glycan alters the accessibility or stability not only of the CD4bs but also of the coreceptor binding site.

TABLE 4

Neutralization sensitivity of WT and N7 mutant pseudotyped viruses to HIV-positive serum and monoclonal antibodies targeting V1-V2, V3, mannan, and MPER epitopes

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a

 See footnote a to Table 2.

 

 

b See footnote b to Table 2.

 

 

c See footnote c to Table 2.

 

 

 

 

 

Effect of the N7 glycan on neutralization sensitivity to monoclonal antibodies directed to quaternary neutralizing epitopes in V2.

 

Since the N7 glycan is located at the C-terminal stem of the V2 loop, we examined if the removal of the N7 glycan would affect virus sensitivity to monoclonal antibodies PG9 and PG16, which recognize a glycan-dependent, quaternary neutralizing epitope in V2 (70). Most of the isolates in the test panel lack the N160 glycan recognized by PG9 and PG16 and are resistant to neutralization by these antibodies (Fig. 2). Therefore, we focused on isolates that are sensitive to PG9 and PG16. Results in Table 4 show that the N7 glycan also affected the sensitivity of HIV-1 to these monoclonal antibodies, but in an isolate-dependent manner. PVO.4 showed increased sensitivity to PG9 and PG16 when the N7 glycan was absent, whereas the other N7 mutant viruses became more resistant. Interestingly, the introduction of a PNLG site upstream of the N7 site in ADA N197S restored sensitivity to PG9 and partially so to PG16, again indicating that a glycan at or around the N7 glycan site may play a role maintaining the quaternary structure recognized by PG9 and PG16. This isolate-dependent effect of the N7 glycan on neutralization sensitivity to PG9 and PG16 is in contrast to its effect on the sensitivity to CD4bs and V3-directed neutralizing antibodies, where an increased sensitivity was observed in all isolates tested. In this context, it is of interest to note that the panel of isolates tested have various V2 loop sequences, amino acid lengths, and numbers of glycosylation sites, including the presence or absence of N160 (Fig. 2), which is a part of the epitope targeted by PG9 and PG16.

 

 

In order to determine if the effect of the N7 glycan on sensitivity to V2-specific antibodies is limited to those isolates that maintain the N160 PNLG, we examined WT and N7 mutant Env from SF162 and JR-FL for reactivity to monoclonal antibody 697-30D, which also recognizes a conformation-dependent epitope in V2 but does not rely upon the quaternary form of Env or the presence of the N160 glycan (Fig. 5) (47). The binding of 697-30D to WT and mutant Env was determined by antigen capture assays using three forms of the Env that differ in their conformational and oligomeric structures: monomeric gp120s and proteins from lysed pseudovirus and whole pseudovirus, with the last exhibiting the most sterically constrained forms of Env (71,–75). Binding of 2G12 and HIV-positive serum was detected with all three forms of Env tested (data not shown) and was therefore used as positive controls. Vaccinia virus-infected cell lysate was included as a negative control. All values described were normalized to binding by HIV-positive serum and were expressed as percentages of binding to the WT Env

 

 

.

FIG 5

Effect of N7 glycan on the binding of a monoclonal antibody recognizing a conformation-dependent epitope in the V2 loop. Binding of monoclonal antibody 697-30D to the WT or N7 mutant forms of SF162 (A) or JR-FL (B) Env was measured by antigen capture assays. Three different forms of Env were tested: (i) whole virions pseudotyped with the indicated Env, (ii) pseudovirions lysed with Triton X-100, or (iii) gp120 expressed in recombinant vaccinia virus-infected cells (61). Binding to 697-30D was normalized to binding obtained with HIV-positive serum, and values obtained with the N7-deglycosylated Env (red bars) were expressed as a percentage of the values obtained with the N7-glycosylated Env (blue bars). The results represent the averages and standard deviations obtained from three individual experiments performed in duplicates. P values were calculated by the Wilcoxon matched-pairs signed-rank test (**, P ≤ 0.01).

N7-deglycosylated SF162 Env from lysed virions and monomeric gp120s showed significantly reduced binding to 697-30D compared to that of their glycosylated counterparts (P ≤ 0.01) (Fig. 5A). However, there was no difference in 697-30D binding between glycosylated and deglycosylated Env when whole SF162 virions were used, indicating that the N7 glycan may alter the conformation of the V2 loop in the less constrained gp120 but not in the trimeric form on whole virions where there is reduced local flexibility. However, no differences were observed between N7-glycosylated or deglycosylated forms of JR-FL Env, as presented in gp120, whole virions, or lysed virions (Fig. 5B), possibly as a result of the more constrained nature of the V2 loop in JR-FL compared to that in SF162. Again, these findings support the notion that the N7 glycan affects the antigenicity of the V2 loop in an isolate-specific manner.

 

Effect of the N7 glycan on neutralization sensitivity to monoclonal antibodies directed to epitopes in the mannan cluster and the MPER.

To examine the effect of the N7 glycan on the antigenicity of distal parts of the Env, we tested the sensitivity of WT and N7 mutant viruses to mannan-dependent and MPER-specific antibodies, 2G12 and 4E10, respectively (34,–36). There was little to no change in 2G12 or 4E10 sensitivity when the N7 glycan was removed (Table 4). While QA255, 89.6, ADA, and LN40 showed ∼3-fold sensitivity difference to 2G12 and PVO.4 and SF162 exhibited a modest (∼4-fold) increase in sensitivity to 4E10 when the N7 glycan was removed, other viruses demonstrated no change. These data indicate that the N7 glycan plays less of a role in these distal epitopes than in the CD4bs and V2 and V3 loops.

 

The N7 glycan modulates virus sensitivity to antibodies from HIV-positive individuals.

The highly conserved nature of the N7 glycan indicates an important role for virus survival. To demonstrate this potential role, we examined the sensitivity of the WT and N7 mutant viruses in the panel of HIV-1 isolates against pooled serum samples from HIV-positive individuals. As expected, there was a wide spectrum of neutralization sensitivities among the viruses tested against the clade B and clade C serum pools (Table 4). However, most of the viruses tested, regardless of their neutralization sensitivities, showed 3- to 60-fold-increased sensitivity to either the clade B or clade C HIV-positive pooled sera when the N7 glycan was absent, consistent with a conserved role of the N7 glycan in the evasion of host neutralizing antibody responses. The only exception was a clade A isolate, QA255, for which the N7-glycosylated form was highly resistant to the clade B serum used (1:20), making it difficult to determine any increase in sensitivity for the N7-deglycosylated virus.

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DISCUSSION

Glycans on HIV-1 envelope proteins have been shown or postulated to play an important and multifaceted role in viral replication, including structural integrity of the virus, coreceptor usage, evasion of host immune responses, and as targets for bNAbs (21, 23, 57, 70, 76). Here, we report a conserved role for the N-linked glycan at amino acid position 197 (N7) in modulating the antigenicity of the CD4bs and epitopes in the V3 loop, a major determinant for coreceptor usage. This role is conserved among a panel of viruses tested, including isolates of different clades, tissue origins, coreceptor usages, and neutralization sensitivities (Tables 2 and ​and4).4). The N7 glycan is also unique among PNLGs in the conserved regions of HIV-1 in modulating the sensitivity to CD4bs-directed neutralizing antibodies while maintaining the integrity of distal epitopes (2G12 and MPER). The increased sensitivity of N7 mutants was not due to a decrease in viral infectivity (Fig. 3) or to any defect in the ability of the N7 mutant gp120 to bind the CD4 receptor (Table 3). The observation that N7 mutants also showed increased sensitivity to pooled HIV-positive serum is consistent with a role for the N7 glycan in viral evasion from the host immune responses in vivo (Table 4).

It is of interest that the absence of the N7 glycan in CNS versus blood or lymph node isolates resulted in differential sensitivities to sCD4. While the blood and lymph node isolates demonstrated increased sensitivity to all CD4bs-directed antibodies when the N7 glycan was absent, the same mutation in CNS isolates showed no change in sensitivity to sCD4 (Table 2). This differential sensitivity may be derived from the immune-privileged environment from which CNS isolates were obtained; i.e., there is little selective pressure for CNS isolates to evade antibodies against the CD4bs (77, 78). CNS isolates typically display a higher avidity for CD4, requiring lower levels to infect cells, and are more resistant to blocking by anti-CD4 antibodies than spleen-derived envelopes (79). Interestingly, the V1-V2, V3, and C2 regions, which include the N7 glycan site, determine the low CD4 dependence and high avidity for CD4 in CNS isolates (80). These factors may contribute to the unchanging sensitivity of CNS isolates to sCD4 when the N7 glycan is removed. However, removal of the N7 glycan did result in increased sensitivity of CNS viruses to other CD4bs-directed antibodies, e.g., VRC01 or IgG1b12, and to V3-directed antibody 447-52D (Tables 2 and ​and4),4), thus conforming to the conserved role of the N7 glycan masking these conserved epitopes.

While the role we ascribe to the N7 glycan is through inference by site-specific mutation with a conserved amino acid substitution (N to Q) at the PNLG sequon, several observations support our notion that the effect of the N7 mutant is due not to the amino acid substitution per se but rather to the absence of the glycan at this sequon. As shown in Table 2, the N197Q and N197S mutants resulted in similar changes in the sensitivity of LN40 to sCD4- and CD4bs-specific antibodies VRC01 and IgG1b12. Interestingly, the same amino acid substitutions in ADA resulted in divergent phenotypes, with the N197S mutant exhibiting a sensitivity to VRC01 comparable to that of the WT virus and increased resistance to IgG1b12, while the N197Q mutant showed increased sensitivity to these antibodies. It is noteworthy that the replacement at amino acid position 197 with S resulted in the creation of a new upstream PNLG sequon at N195 (Fig. 2). Thus, we interpret the divergent phenotype of the N197Q and N197S mutants as evidence supporting a conserved role for an N-linked glycan at the base of the V2 loop: either the N7 glycan in the case of the WT ADA or the newly created glycan at N195 in the case of N197S. The differences in the apparent molecular weights of WT and N7 mutant Env expressed in recombinant vaccinia viruses also support the occupancy of the N7 PNLG in all the isolates tested (data not shown).

Although the N7 glycan modulates the receptor and coreceptor binding site in a conserved manner, its effect on the ability of the Env to mediate CD4-independent entry is specific to the isolate tested and the specific amino acid change introduced at the PNLG site. Data from this and previous studies showed ADA N197S and 89.6 as the only two isolates that display enhanced ability to mediate CD4-independent entry when the N7 glycan is absent (Fig. 4) (21, 22). In these cases, the enhanced ability to mediate CD4-independent entry correlated with increased sensitivity to CD4i antibody 17b (Table 2) (22). However, for other isolates tested, the presence or absence of the N7 glycan does not affect 17b sensitivity, even though increased susceptibility to a V3 antibody, 447-52D, was observed (Table 4). Furthermore, as in the case of ADA, the creation of an upstream PNLG sequon in the N197S mutant resulted in the acquisition of CD4-independent phenotype, whereas the absence of the N7 glycan in N197Q retained CD4 dependence (Fig. 4). Thus, even though the N7 glycan modulates the access or stability of epitopes in the CD4bs and coreceptor binding sites, neither the N7 glycan nor susceptibility to 17b per se is predictive of CD4 independence in an isolate-dependent or sequence-specific manner.

The N7 glycan also modulates the sensitivity of virus to neutralizing antibodies recognizing a quaternary structure in V1-V2. Interestingly, this effect appears to be isolate dependent (Table 4) (81). PVO.4 was the only isolate that demonstrated increased sensitivity to PG9 and PG16 antibodies when the N7 glycan was removed, while the same glycan mutant in every other virus tested showed increased resistance. Since PVO.4 has the longest V2 loop length and the highest number of potential N-linked glycan sites, we speculate that PVO.4 may have a higher degree of structural constraint in the V2 loop than other isolates tested. If so, the conformation-dependent epitope in V2 recognized by PG9 and PG16 may be better preserved in PVO.4 than in the other isolates. Thus, when the N7 glycan is removed, the better-preserved PG9 and PG16 epitope in PVO.4 Env may be better exposed (and thus the higher sensitivity to these MAb), whereas the greater flexibility in other isolates may result in the loss of this epitope (and thus greater resistance). Furthermore, we note that the isolate-dependent effect of the N7 glycan on the epitopes recognized by V2-specific MAb is not dependent on the presence or the nature of the N160 glycan in the isolates tested. This is because the N7 glycan also affects Env binding to a V2-specific, conformation-dependent antibody, 697-30D, the epitope of which is not dependent on the N160 glycan (Fig. 5). Thus, the isolate-dependent effect of the N7 glycan on V2 antigenicity is likely mediated through its effect on the positioning or the conformation of the V1-V2 loop.

Currently, we do not have a clear understanding of the basis of the effect of the N7 glycan reported in this study. As shown in Table 3, the presence or absence of the N7 glycan does not affect the affinity of gp120 to CD4-IgG2. This observation is consistent with previous publications demonstrating that the CD4 binding site on gp120 is not modified by carbohydrates (49, 82). Others have demonstrated that the N7 glycan may help to stabilize the native Env by occluding the V3 loop, shielding V3 epitopes, and inhibiting its premature release prior to CD4 binding (68, 69). These findings, together with our data reported here, support the possibility that the biological property of the N7 glycan may only be discernible in the trimeric form of Env. Furthermore, we have little information on the nature of the glycans on the N7 site and how that may affect its role in different isolates. Further studies will be needed to provide a better understanding of the structural and biophysical basis of our observations.

We report here the conserved phenotype of an N-linked glycan in masking epitopes in the CD4bs and coreceptor binding sites, epitopes targeted by some of the most potent and broadly reactive neutralizing antibodies. Because glycans on HIV-1 play an important role in the structure and function of Env (63, 83, 84) and because multiple studies have shown that carbohydrates modulate Env antigenicity (21, 23, 57, 70, 76, 85,–90), glycan modification may represent a rational approach for immunogen design. Although multiple studies have shown little or negative impact of glycan modifications on Env immunogenicity (91,–95), we previously demonstrated that removal of specific glycans may enhance the ability of HIV Env to elicit neutralizing antibody responses (21). Similar results were also obtained by the removal of specific N-linked glycans proximal to the CD4 binding region (23). Mutant Env with specific glycans removed also binds to the unmutated common ancestors of broadly neutralizing MAbs better than glycosylated versions (88, 96). Furthermore, glycan modification may play a role in modifying the immunogenicity of other vaccine candidates, including those for hemorrhagic fever (97) and feline immunodeficiency virus (98). Taken together, our finding of the conserved nature of the N7 glycan in masking the receptor and coreceptor binding sites of HIV-1 Env may inform the design of immunogens more capable of eliciting bNAbs against these important targets.

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ACKNOWLEDGMENTS

We thank Wenjin Guo, Patricia Firpo, Thaddeus Davenport, and Miklos Guttman for expert advice; Taryn Urion and Jennifer McKenna for technical assistance; Cecilia Cheng-Mayer, Paul Clapham, Julie Overbaugh, James Robinson, Ruth Ruprecht, Joseph Sodroski, Leonidas Stamatatos, Charles Wood, Susan Zolla-Pazner, and the NIH AIDS Research and Reference Reagent Program for providing monoclonal antibodies and reagents; and Shan Lu and Kelly Lee for helpful discussions.

This study was supported in part by NIH grants R01 AI076170, P01 AI082274, and P51 OD010425 and Bill and Melinda Gates Foundation award OPP1033102.

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FUNDING STATEMENT

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication

 

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REFERENCES

  1. Pegu AYZ, Chen X, Todd JP, McKee K, Rao S, Mascola J, Nabel G. 2011. VRC01 provides sterilizing protection to non human primates from mucosal SHIV challenges. J Immunol 186:11. [Google Scholar]
  2. Ruprecht RM. 2009. Passive immunization with human neutralizing monoclonal antibodies against HIV-1 in macaque models: experimental approaches. Methods Mol Biol 525:559–566, xiv. doi:10.1007/978-1-59745-554-1_31. [PubMed] [CrossRef] [Google Scholar]
  3. Ruprecht RM, Ferrantelli F, Kitabwalla M, Xu W, McClure HM. 2003. Antibody protection: passive immunization of neonates against oral AIDS virus challenge. Vaccine 21:3370–3373. doi:10.1016/S0264-410X(03)00335-9. [PubMed] [CrossRef] [Google Scholar]
  4. Haigwood NL, Montefiori DC, Sutton WF, McClure J, Watson AJ, Voss G, Hirsch VM, Richardson BA, Letvin NL, Hu SL, Johnson PR. 2004. Passive immunotherapy in simian immunodeficiency virus-infected macaques accelerates the development of neutralizing antibodies. J Virol 78:5983–5995. doi:10.1128/JVI.78.11.5983-5995.2004. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
  5. Hessell AJ, Rakasz EG, Tehrani DM, Huber M, Weisgrau KL, Landucci G, Forthal DN, Koff WC, Poignard P, Watkins DI, Burton DR. 2010. Broadly neutralizing monoclonal antibodies 2F5 and 4E10 directed against the human immunodeficiency virus type 1 gp41 membrane-proximal external region protect against mucosal challenge by simian-human immunodeficiency virus SHIVBa-L. J Virol 84:1302–1313. doi:10.1128/JVI.01272-09. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
  6. Burton DR, Poignard P, Stanfield RL, Wilson IA. 2012. Broadly neutralizing antibodies present new prospects to counter highly antigenically diverse viruses. Science 337:183–186. doi:10.1126/science.1225416. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
  7. Kwong PD, Mascola JR. 2012. Human antibodies that neutralize HIV-1: identification, structures, and B cell ontogenies. Immunity 37:412–425. doi:10.1016/j.immuni.2012.08.012. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
  8. Saunders KO, Rudicell RS, Nabel GJ. 2012. The design and evaluation of HIV-1 vaccines. AIDS 26:1293–1302. doi:10.1097/QAD.0b013e32835474d2. [PubMed] [CrossRef] [Google Scholar]
  9. Haynes BF, Kelsoe G, Harrison SC, Kepler TB. 2012. B-cell-lineage immunogen design in vaccine development with HIV-1 as a case study. Nat Biotechnol 30:423–433. doi:10.1038/nbt.2197. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
  10. Mikell I, Sather DN, Kalams SA, Altfeld M, Alter G, Stamatatos L. 2011. Characteristics of the earliest cross-neutralizing antibody response to HIV-1. PLoS Pathog 7:e1001251. doi:10.1371/journal.ppat.1001251. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
  11. Mascola JR, Montefiori DC. 2010. The role of antibodies in HIV vaccines. Annu Rev Immunol 28:413–444. doi:10.1146/annurev-immunol-030409-101256. [PubMed] [CrossRef] [Google Scholar]
  12. Blattner C, Lee JH, Sliepen K, Derking R, Falkowska E, de la Pena AT, Cupo A, Julien JP, van Gils M, Lee PS, Peng W, Paulson JC, Poignard P, Burton DR, Moore JP, Sanders RW, Wilson IA, Ward AB. 2014. Structural delineation of a quaternary, cleavage-dependent epitope at the gp41-gp120 interface on intact HIV-1 Env trimers. Immunity 40:669–680. doi:10.1016/j.immuni.2014.04.008. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
  13. Huang J, Kang BH, Pancera M, Lee JH, Tong T, Feng Y, Imamichi H, Georgiev IS, Chuang GY, Druz A, Doria-Rose NA, Laub L, Sliepen K, van Gils MJ, de la Pena AT, Derking R, Klasse PJ, Migueles SA, Bailer RT, Alam M, Pugach P, Haynes BF, Wyatt RT, Sanders RW, Binley JM, Ward AB, Mascola JR, Kwong PD, Connors M. 2014. Broad and potent HIV-1 neutralization by a human antibody that binds the gp41-gp120 interface. Nature 515:138–142. doi:10.1038/nature13601. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
  14. Overbaugh J, Rudensey LM. 1992. Alterations in potential sites for glycosylation predominate during evolution of the simian immunodeficiency virus envelope gene in macaques. J Virol 66:5937–5948. [PMC free article] [PubMed] [Google Scholar]
  15. Wei X, Decker JM, Wang S, Hui H, Kappes JC, Wu X, Salazar-Gonzalez JF, Salazar MG, Kilby JM, Saag MS, Komarova NL, Nowak MA, Hahn BH, Kwong PD, Shaw GM. 2003. Antibody neutralization and escape by HIV-1. Nature 422:307–312. doi:10.1038/nature01470. [PubMed] [CrossRef] [Google Scholar]
  16. Derdeyn CA, Decker JM, Bibollet-Ruche F, Mokili JL, Muldoon M, Denham SA, Heil ML, Kasolo F, Musonda R, Hahn BH, Shaw GM, Korber BT, Allen S, Hunter E. 2004. Envelope-constrained neutralization-sensitive HIV-1 after heterosexual transmission. Science 303:2019–2022. doi:10.1126/science.1093137. [PubMed] [CrossRef] [Google Scholar]
  17. Sagar M, Wu X, Lee S, Overbaugh J. 2006. Human immunodeficiency virus type 1 V1-V2 envelope loop sequences expand and add glycosylation sites over the course of infection, and these modifications affect antibody neutralization sensitivity. J Virol 80:9586–9598. doi:10.1128/JVI.00141-06. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
  18. Reitter JN, Means RE, Desrosiers RC. 1998. A role for carbohydrates in immune evasion in AIDS. Nat Med 4:679–684. doi:10.1038/nm0698-679. [PubMed] [CrossRef] [Google Scholar]
  19. Zhang M, Gaschen B, Blay W, Foley B, Haigwood N, Kuiken C, Korber B. 2004. Tracking global patterns of N-linked glycosylation site variation in highly variable viral glycoproteins: HIV, SIV, and HCV envelopes and influenza hemagglutinin. Glycobiology 14:1229–1246. doi:10.1093/glycob/cwh106. [PubMed] [CrossRef] [Google Scholar]
  20. Frost SDW, Wrin T, Smith DM, Pond SLK, Liu Y, Paxinos E, Chappey C, Galovich J, Beauchaine J, Petropoulos CJ, Little SJ, Richman DD. 2005. Neutralizing antibody responses drive the evolution of human immunodeficiency virus type 1 envelope during recent HIV infection. Proc Natl Acad Sci U S A 102:18514–18519. doi:10.1073/pnas.0504658102. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
  21. Li Y, Cleveland B, Klots I, Travis B, Richardson BA, Anderson D, Montefiori D, Polacino P, Hu SL. 2008. Removal of a single N-linked glycan in human immunodeficiency virus type 1 gp120 results in an enhanced ability to induce neutralizing antibody responses. J Virol 82:638–651. doi:10.1128/JVI.01691-07. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
  22. Kolchinsky P, Kiprilov E, Bartley P, Rubinstein R, Sodroski J. 2001. Loss of a single N-linked glycan allows CD4-independent human immunodeficiency virus type 1 infection by altering the position of the gp120 V1/V2 variable loops. J Virol 75:3435–3443. doi:10.1128/JVI.75.7.3435-3443.2001. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
  23. Huang X, Jin W, Hu K, Luo S, Du T, Griffin GE, Shattock RJ, Hu Q. 2012. Highly conserved HIV-1 gp120 glycans proximal to CD4-binding region affect viral infectivity and neutralizing antibody induction. Virology 423:97–106. doi:10.1016/j.virol.2011.11.023. [PubMed] [CrossRef] [Google Scholar]
  24. Pikora C, Wittish C, Desrosiers RC. 2005. Identification of two N-linked glycosylation sites within the core of the simian immunodeficiency virus glycoprotein whose removal enhances sensitivity to soluble CD4. J Virol 79:12575–12583. doi:10.1128/JVI.79.19.12575-12583.2005. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
  25. Bosch KA, Rainwater S, Jaoko W, Overbaugh J. 2010. Temporal analysis of HIV envelope sequence evolution and antibody escape in a subtype A-infected individual with a broad neutralizing antibody response. Virology 398:115–124. doi:10.1016/j.virol.2009.11.032. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
  26. Karlsson GB, Halloran M, Li J, Park IW, Gomila R, Reimann KA, Axthelm MK, Iliff SA, Letvin NL, Sodroski J. 1997. Characterization of molecularly cloned simian-human immunodeficiency viruses causing rapid CD4+ lymphocyte depletion in rhesus monkeys. J Virol 71:4218–4225. [PMC free article] [PubMed] [Google Scholar]
  27. Kolchinsky P, Mirzabekov T, Farzan M, Kiprilov E, Cayabyab M, Mooney LJ, Choe H, Sodroski J. 1999. Adaptation of a CCR5-using, primary human immunodeficiency virus type 1 isolate for CD4-independent replication. J Virol 73:8120–8126. [PMC free article] [PubMed] [Google Scholar]
  28. Peters PJ, Bhattacharya J, Hibbitts S, Dittmar MT, Simmons G, Bell J, Simmonds P, Clapham PR. 2004. Biological analysis of human immunodeficiency virus type 1 R5 envelopes amplified from brain and lymph node tissues of AIDS patients with neuropathology reveals two distinct tropism phenotypes and identifies envelopes in the brain that confer an enhanced tropism and fusigenicity for macrophages. J Virol 78:6915–6926. doi:10.1128/JVI.78.13.6915-6926.2004. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
  29. Koyanagi Y, Miles S, Mitsuyasu RT, Merrill JE, Vinters HV, Chen IS. 1987. Dual infection of the central nervous system by AIDS viruses with distinct cellular tropisms. Science 236:819–822. doi:10.1126/science.3646751. [PubMed] [CrossRef] [Google Scholar]
  30. Cheng-Mayer C, Liu R, Landau NR, Stamatatos L. 1997. Macrophage tropism of human immunodeficiency virus type 1 and utilization of the CC-CKR5 coreceptor. J Virol 71:1657–1661. [PMC free article] [PubMed] [Google Scholar]
  31. Grisson RD, Chenine AL, Yeh LY, He J, Wood C, Bhat GJ, Xu W, Kankasa C, Ruprecht RM. 2004. Infectious molecular clone of a recently transmitted pediatric human immunodeficiency virus clade C isolate from Africa: evidence of intraclade recombination. J Virol 78:14066–14069. doi:10.1128/JVI.78.24.14066-14069.2004. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
  32. Li M, Gao F, Mascola JR, Stamatatos L, Polonis VR, Koutsoukos M, Voss G, Goepfert P, Gilbert P, Greene KM, Bilska M, Kothe DL, Salazar-Gonzalez JF, Wei X, Decker JM, Hahn BH, Montefiori DC. 2005. Human immunodeficiency virus type 1 env clones from acute and early subtype B infections for standardized assessments of vaccine-elicited neutralizing antibodies. J Virol 79:10108–10125. doi:10.1128/JVI.79.16.10108-10125.2005. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
  33. Gao F, Morrison SG, Robertson DL, Thornton CL, Craig S, Karlsson G, Sodroski J, Morgado M, Galvao-Castro B, von Briesen H, Beddows S, Weber J, Sharp PM, Shaw GM, Hahn BH. 1996. Molecular cloning and analysis of functional envelope genes from human immunodeficiency virus type 1 sequence subtypes A through G. The WHO and NIAID Networks for HIV Isolation and Characterization. J Virol 70:1651–1667. [PMC free article] [PubMed] [Google Scholar]
  34. Stiegler G, Kunert R, Purtscher M, Wolbank S, Voglauer R, Steindl F, Katinger H. 2001. A potent cross-clade neutralizing human monoclonal antibody against a novel epitope on gp41 of human immunodeficiency virus type 1. AIDS Res Hum Retroviruses 17:1757–1765. doi:10.1089/08892220152741450. [PubMed] [CrossRef] [Google Scholar]
  35. Buchacher A, Predl R, Strutzenberger K, Steinfellner W, Trkola A, Purtscher M, Gruber G, Tauer C, Steindl F, Jungbauer A, Katinger H. 1994. Generation of human monoclonal antibodies against HIV-1 proteins; electrofusion and Epstein-Barr virus transformation for peripheral blood lymphocyte immortalization. AIDS Res Hum Retroviruses 10:359–369. doi:10.1089/aid.1994.10.359. [PubMed] [CrossRef] [Google Scholar]
  36. Trkola A, Purtscher M, Muster T, Ballaun C, Buchacher A, Sullivan N, Srinivasan K, Sodroski J, Moore JP, Katinger H. 1996. Human monoclonal antibody 2G12 defines a distinctive neutralization epitope on the gp120 glycoprotein of human immunodeficiency virus type 1. J Virol 70:1100–1108. [PMC free article] [PubMed] [Google Scholar]
  37. Barbas CF III, Bjorling E, Chiodi F, Dunlop N, Cababa D, Jones TM, Zebedee SL, Persson MA, Nara PL, Norrby E. 1992. Recombinant human Fab fragments neutralize human type 1 immunodeficiency virus in vitro. Proc Natl Acad Sci U S A 89:9339–9343. doi:10.1073/pnas.89.19.9339. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
  38. Burton DR, Barbas CF III, Persson MA, Koenig S, Chanock RM, Lerner RA. 1991. A large array of human monoclonal antibodies to type 1 human immunodeficiency virus from combinatorial libraries of asymptomatic seropositive individuals. Proc Natl Acad Sci U S A 88:10134–10137. doi:10.1073/pnas.88.22.10134. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
  39. Burton DR, Pyati J, Koduri R, Sharp SJ, Thornton GB, Parren PW, Sawyer LS, Hendry RM, Dunlop N, Nara PL, Lamacchia M, Garratti E, Stiehm ER, Bryson YJ, Cao Y, Moore JP, Ho DD, Barbas CF III. 1994. Efficient neutralization of primary isolates of HIV-1 by a recombinant human monoclonal antibody. Science 266:1024–1027. doi:10.1126/science.7973652. [PubMed] [CrossRef] [Google Scholar]
  40. Roben P, Moore JP, Thali M, Sodroski J, Barbas CF III, Burton DR. 1994. Recognition properties of a panel of human recombinant Fab fragments to the CD4 binding site of gp120 that show differing abilities to neutralize human immunodeficiency virus type 1. J Virol 68:4821–4828. [PMC free article] [PubMed] [Google Scholar]
  41. Cook RF, Berger SL, Rushlow KE, McManus JM, Cook SJ, Harrold S, Raabe ML, Montelaro RC, Issel CJ. 1995. Enhanced sensitivity to neutralizing antibodies in a variant of equine infectious anemia virus is linked to amino acid substitutions in the surface unit envelope glycoprotein. J Virol 69:1493–1499. [PMC free article] [PubMed] [Google Scholar]
  42. Gorny MK, Conley AJ, Karwowska S, Buchbinder A, Xu JY, Emini EA, Koenig S, Zolla-Pazner S. 1992. Neutralization of diverse human immunodeficiency virus type 1 variants by an anti-V3 human monoclonal antibody. J Virol 66:7538–7542. [PMC free article] [PubMed] [Google Scholar]
  43. Gorny MK, VanCott TC, Hioe C, Israel ZR, Michael NL, Conley AJ, Williams C, Kessler JA II, Chigurupati P, Burda S, Zolla-Pazner S. 1997. Human monoclonal antibodies to the V3 loop of HIV-1 with intra- and interclade cross-reactivity. J Immunol 159:5114–5122. [PubMed] [Google Scholar]
  44. Gorny MK, Xu JY, Karwowska S, Buchbinder A, Zolla-Pazner S. 1993. Repertoire of neutralizing human monoclonal antibodies specific for the V3 domain of HIV-1 gp120. J Immunol 150:635–643. [PubMed] [Google Scholar]
  45. Nyambi PN, Gorny MK, Bastiani L, van der Groen G, Williams C, Zolla-Pazner S. 1998. Mapping of epitopes exposed on intact human immunodeficiency virus type 1 (HIV-1) virions: a new strategy for studying the immunologic relatedness of HIV-1. J Virol 72:9384–9391. [PMC free article] [PubMed] [Google Scholar]
  46. Zolla-Pazner S, O'Leary J, Burda S, Gorny MK, Kim M, Mascola J, McCutchan F. 1995. Serotyping of primary human immunodeficiency virus type 1 isolates from diverse geographic locations by flow cytometry. J Virol 69:3807–3815. [PMC free article] [PubMed] [Google Scholar]
  47. Gorny MK, Moore JP, Conley AJ, Karwowska S, Sodroski J, Williams C, Burda S, Boots LJ, Zolla-Pazner S. 1994. Human anti-V2 monoclonal antibody that neutralizes primary but not laboratory isolates of human immunodeficiency virus type 1. J Virol 68:8312–8320. [PMC free article] [PubMed] [Google Scholar]
  48. Israel ZR, Gorny MK, Palmer C, McKeating JA, Zolla-Pazner S. 1997. Prevalence of a V2 epitope in clade B primary isolates and its recognition by sera from HIV-1-infected individuals. AIDS 11:128–130. [PubMed] [Google Scholar]
  49. Kwong PD, Wyatt R, Robinson J, Sweet RW, Sodroski J, Hendrickson WA. 1998. Structure of an HIV gp120 envelope glycoprotein in complex with the CD4 receptor and a neutralizing human antibody. Nature 393:648–659. doi:10.1038/31405. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
  50. Sullivan N, Sun Y, Sattentau Q, Thali M, Wu D, Denisova G, Gershoni J, Robinson J, Moore J, Sodroski J. 1998. CD4-Induced conformational changes in the human immunodeficiency virus type 1 gp120 glycoprotein: consequences for virus entry and neutralization. J Virol 72:4694–4703. [PMC free article] [PubMed] [Google Scholar]
  51. Thali M, Moore JP, Furman C, Charles M, Ho DD, Robinson J, Sodroski J. 1993. Characterization of conserved human immunodeficiency virus type 1 gp120 neutralization epitopes exposed upon gp120-CD4 binding. J Virol 67:3978–3988. [PMC free article] [PubMed] [Google Scholar]
  52. Trkola A, Dragic T, Arthos J, Binley JM, Olson WC, Allaway GP, Cheng-Mayer C, Robinson J, Maddon PJ, Moore JP. 1996. CD4-dependent, antibody-sensitive interactions between HIV-1 and its co-receptor CCR-5. Nature 384:184–187. doi:10.1038/384184a0. [PubMed] [CrossRef] [Google Scholar]
  53. Wyatt R, Kwong PD, Desjardins E, Sweet RW, Robinson J, Hendrickson WA, Sodroski JG. 1998. The antigenic structure of the HIV gp120 envelope glycoprotein. Nature 393:705–711. doi:10.1038/31514. [PubMed] [CrossRef] [Google Scholar]
  54. Wyatt R, Moore J, Accola M, Desjardin E, Robinson J, Sodroski J. 1995. Involvement of the V1/V2 variable loop structure in the exposure of human immunodeficiency virus type 1 gp120 epitopes induced by receptor binding. J Virol 69:5723–5733. [PMC free article] [PubMed] [Google Scholar]
  55. Garlick RL, Kirschner RJ, Eckenrode FM, Tarpley WG, Tomich CS. 1990. Escherichia coli expression, purification, and biological activity of a truncated soluble CD4. AIDS Res Hum Retroviruses 6:465–479. doi:10.1089/aid.1990.6.465. [PubMed] [CrossRef] [Google Scholar]
  56. Wu X, Yang ZY, Li Y, Hogerkorp CM, Schief WR, Seaman MS, Zhou T, Schmidt SD, Wu L, Xu L, Longo NS, McKee K, O'Dell S, Louder MK, Wycuff DL, Feng Y, Nason M, Doria-Rose N, Connors M, Kwong PD, Roederer M, Wyatt RT, Nabel GJ, Mascola JR. 2010. Rational design of envelope identifies broadly neutralizing human monoclonal antibodies to HIV-1. Science 329:856–861. doi:10.1126/science.1187659. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
  57. Walker LM, Huber M, Doores KJ, Falkowska E, Pejchal R, Julien JP, Wang SK, Ramos A, Chan-Hui PY, Moyle M, Mitcham JL, Hammond PW, Olsen OA, Phung P, Fling S, Wong CH, Phogat S, Wrin T, Simek MD, Koff WC, Wilson IA, Burton DR, Poignard P. 2011. Broad neutralization coverage of HIV by multiple highly potent antibodies. Nature 477:466–470. doi:10.1038/nature10373. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
  58. Montefiori DC. 2005. Evaluating neutralizing antibodies against HIV, SIV, and SHIV in luciferase reporter gene assays. Curr Protoc Immunol Chapter 12:Unit 12.11. [PubMed] [Google Scholar]
  59. Abdiche Y, Malashock D, Pinkerton A, Pons J. 2008. Determining kinetics and affinities of protein interactions using a parallel real-time label-free biosensor, the Octet. Anal Biochem 377:209–217. doi:10.1016/j.ab.2008.03.035. [PubMed] [CrossRef] [Google Scholar]
  60. Mayr LM, Cohen S, Spurrier B, Kong XP, Zolla-Pazner S. 2013. Epitope mapping of conformational V2-specific anti-HIV human monoclonal antibodies reveals an immunodominant site in V2. PLoS One 8:e70859. doi:10.1371/journal.pone.0070859. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
  61. Guo W, Cleveland B, Davenport TM, Lee KK, Hu SL. 2013. Purification of recombinant vaccinia virus-expressed monomeric HIV-1 gp120 to apparent homogeneity. Protein Expr Purif 90:34–39. doi:10.1016/j.pep.2013.04.009. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
  62. Zhou T, Georgiev I, Wu X, Yang ZY, Dai K, Finzi A, Kwon YD, Scheid JF, Shi W, Xu L, Yang Y, Zhu J, Nussenzweig MC, Sodroski J, Shapiro L, Nabel GJ, Mascola JR, Kwong PD. 2010. Structural basis for broad and potent neutralization of HIV-1 by antibody VRC01. Science 329:811–817. doi:10.1126/science.1192819. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
  63. Li Y, Rey-Cuille MA, Hu SL. 2001. N-linked glycosylation in the V3 region of HIV type 1 surface antigen modulates coreceptor usage in viral infection. AIDS Res Hum Retroviruses 17:1473–1479. doi:10.1089/08892220152644179. [PubMed] [CrossRef] [Google Scholar]
  64. Brighty DW, Rosenberg M, Chen IS, Ivey-Hoyle M. 1991. Envelope proteins from clinical isolates of human immunodeficiency virus type 1 that are refractory to neutralization by soluble CD4 possess high affinity for the CD4 receptor. Proc Natl Acad Sci U S A 88:7802–7805. doi:10.1073/pnas.88.17.7802. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
  65. Ivey-Hoyle M, Culp JS, Chaikin MA, Hellmig BD, Matthews TJ, Sweet RW, Rosenberg M. 1991. Envelope glycoproteins from biologically diverse isolates of immunodeficiency viruses have widely different affinities for CD4. Proc Natl Acad Sci U S A 88:512–516. doi:10.1073/pnas.88.2.512. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
  66. Mulligan MJ, Ritter GD Jr, Chaikin MA, Yamshchikov GV, Kumar P, Hahn BH, Sweet RW, Compans RW. 1992. Human immunodeficiency virus type 2 envelope glycoprotein: differential CD4 interactions of soluble gp120 versus the assembled envelope complex. Virology 187:233–241. doi:10.1016/0042-6822(92)90311-C. [PubMed] [CrossRef] [Google Scholar]
  67. Seaman MS, Janes H, Hawkins N, Grandpre LE, Devoy C, Giri A, Coffey RT, Harris L, Wood B, Daniels MG, Bhattacharya T, Lapedes A, Polonis VR, McCutchan FE, Gilbert PB, Self SG, Korber BT, Montefiori DC, Mascola JR. 2010. Tiered categorization of a diverse panel of HIV-1 Env pseudoviruses for assessment of neutralizing antibodies. J Virol 84:1439–1452. doi:10.1128/JVI.02108-09. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
  68. Lyumkis D, Julien JP, de Val N, Cupo A, Potter CS, Klasse PJ, Burton DR, Sanders RW, Moore JP, Carragher B, Wilson IA, Ward AB. 2013. Cryo-EM structure of a fully glycosylated soluble cleaved HIV-1 envelope trimer. Science 342:1484–1490. doi:10.1126/science.1245627. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
  69. Julien JP, Cupo A, Sok D, Stanfield RL, Lyumkis D, Deller MC, Klasse PJ, Burton DR, Sanders RW, Moore JP, Ward AB, Wilson IA. 2013. Crystal structure of a soluble cleaved HIV-1 envelope trimer. Science 342:1477–1483. doi:10.1126/science.1245625. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
  70. Walker LM, Phogat SK, Chan-Hui PY, Wagner D, Phung P, Goss JL, Wrin T, Simek MD, Fling S, Mitcham JL, Lehrman JK, Priddy FH, Olsen OA, Frey SM, Hammond PW, Kaminsky S, Zamb T, Moyle M, Koff WC, Poignard P, Burton DR. 2009. Broad and potent neutralizing antibodies from an African donor reveal a new HIV-1 vaccine target. Science 326:285–289. doi:10.1126/science.1178746. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
  71. Poignard P, Saphire EO, Parren PW, Burton DR. 2001. gp120: biologic aspects of structural features. Annu Rev Immunol 19:253–274. doi:10.1146/annurev.immunol.19.1.253. [PubMed] [CrossRef] [Google Scholar]
  72. Weiss CD, Levy JA, White JM. 1990. Oligomeric organization of gp120 on infectious human immunodeficiency virus type 1 particles. J Virol 64:5674–5677. [PMC free article] [PubMed] [Google Scholar]
  73. Weissenhorn W, Dessen A, Harrison SC, Skehel JJ, Wiley DC. 1997. Atomic structure of the ectodomain from HIV-1 gp41. Nature 387:426–430. doi:10.1038/387426a0. [PubMed] [CrossRef] [Google Scholar]
  74. Labrijn AF, Poignard P, Raja A, Zwick MB, Delgado K, Franti M, Binley J, Vivona V, Grundner C, Huang CC, Venturi M, Petropoulos CJ, Wrin T, Dimitrov DS, Robinson J, Kwong PD, Wyatt RT, Sodroski J, Burton DR. 2003. Access of antibody molecules to the conserved coreceptor binding site on glycoprotein gp120 is sterically restricted on primary human immunodeficiency virus type 1. J Virol 77:10557–10565. doi:10.1128/JVI.77.19.10557-10565.2003. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
  75. Chen L, Kwon YD, Zhou T, Wu X, O'Dell S, Cavacini L, Hessell AJ, Pancera M, Tang M, Xu L, Yang ZY, Zhang MY, Arthos J, Burton DR, Dimitrov DS, Nabel GJ, Posner MR, Sodroski J, Wyatt R, Mascola JR, Kwong PD. 2009. Structural basis of immune evasion at the site of CD4 attachment on HIV-1 gp120. Science 326:1123–1127. doi:10.1126/science.1175868. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
  76. O'Rourke SM, Schweighardt B, Phung P, Mesa KA, Vollrath AL, Tatsuno GP, To B, Sinangil F, Limoli K, Wrin T, Berman PW. 2012. Sequences in glycoprotein gp41, the CD4 binding site, and the V2 domain regulate sensitivity and resistance of HIV-1 to broadly neutralizing antibodies. J Virol 86:12105–12114. doi:10.1128/JVI.01352-12. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
  77. Hanly A, Petito CK. 1998. HLA-DR-positive dendritic cells of the normal human choroid plexus: a potential reservoir of HIV in the central nervous system. Hum Pathol 29:88–93. doi:10.1016/S0046-8177(98)90395-1. [PubMed] [CrossRef] [Google Scholar]
  78. Muldoon LL, Alvarez JI, Begley DJ, Boado RJ, Del Zoppo GJ, Doolittle ND, Engelhardt B, Hallenbeck JM, Lonser RR, Ohlfest JR, Prat A, Scarpa M, Smeyne RJ, Drewes LR, Neuwelt EA. 2013. Immunologic privilege in the central nervous system and the blood-brain barrier. J Cereb Blood Flow Metab 33:13–21. doi:10.1038/jcbfm.2012.153. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
  79. Martín-García J, Cao W, Varela-Rohena A, Plassmeyer ML, Gonzalez-Scarano F. 2006. HIV-1 tropism for the central nervous system: brain-derived envelope glycoproteins with lower CD4 dependence and reduced sensitivity to a fusion inhibitor. Virology 346:169–179. doi:10.1016/j.virol.2005.10.031. [PubMed] [CrossRef] [Google Scholar]
  80. Rossi F, Querido B, Nimmagadda M, Cocklin S, Navas-Martin S, Martin-Garcia J. 2008. The V1-V3 region of a brain-derived HIV-1 envelope glycoprotein determines macrophage tropism, low CD4 dependence, increased fusogenicity and altered sensitivity to entry inhibitors. Retrovirology 5:89. doi:10.1186/1742-4690-5-89. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
  81. Gorny MK, Stamatatos L, Volsky B, Revesz K, Williams C, Wang XH, Cohen S, Staudinger R, Zolla-Pazner S. 2005. Identification of a new quaternary neutralizing epitope on human immunodeficiency virus type 1 virus particles. J Virol 79:5232–5237. doi:10.1128/JVI.79.8.5232-5237.2005. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
  82. Myszka DG, Sweet RW, Hensley P, Brigham-Burke M, Kwong PD, Hendrickson WA, Wyatt R, Sodroski J, Doyle ML. 2000. Energetics of the HIV gp120-CD4 binding reaction. Proc Natl Acad Sci U S A 97:9026–9031. doi:10.1073/pnas.97.16.9026. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
  83. Haggerty S, Dempsey MP, Bukrinsky MI, Guo L, Stevenson M. 1991. Posttranslational modifications within the HIV-1 envelope glycoprotein which restrict virus assembly and CD4-dependent infection. AIDS Res Hum Retroviruses 7:501–510. doi:10.1089/aid.1991.7.501. [PubMed] [CrossRef] [Google Scholar]
  84. Hansen JE, Witzke NM, Nielsen C, Mathiesen LR, Teglbjaerg LS, Nielsen CM, Nielsen JO. 1990. Derivatives of amphotericin inhibit infection with human immunodeficiency virus in vitro by different modes of action. Antiviral Res 14:149–159. doi:10.1016/0166-3542(90)90031-2. [PubMed] [CrossRef] [Google Scholar]
  85. Back NK, Smit L, De Jong JJ, Keulen W, Schutten M, Goudsmit J, Tersmette M. 1994. An N-glycan within the human immunodeficiency virus type 1 gp120 V3 loop affects virus neutralization. Virology 199:431–438. doi:10.1006/viro.1994.1141. [PubMed] [CrossRef] [Google Scholar]
  86. Clevestig P, Pramanik L, Leitner T, Ehrnst A. 2006. CCR5 use by human immunodeficiency virus type 1 is associated closely with the gp120 V3 loop N-linked glycosylation site. J Gen Virol 87:607–612. doi:10.1099/vir.0.81510-0. [PubMed] [CrossRef] [Google Scholar]
  87. Kang SM, Quan FS, Huang C, Guo L, Ye L, Yang C, Compans RW. 2005. Modified HIV envelope proteins with enhanced binding to neutralizing monoclonal antibodies. Virology 331:20–32. doi:10.1016/j.virol.2004.10.005. [PubMed] [CrossRef] [Google Scholar]
  88. Ma BJ, Alam SM, Go EP, Lu X, Desaire H, Tomaras GD, Bowman C, Sutherland LL, Scearce RM, Santra S, Letvin NL, Kepler TB, Liao HX, Haynes BF. 2011. Envelope deglycosylation enhances antigenicity of HIV-1 gp41 epitopes for both broad neutralizing antibodies and their unmutated ancestor antibodies. PLoS Pathog 7:e1002200. doi:10.1371/journal.ppat.1002200. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
  89. McCaffrey RA, Saunders C, Hensel M, Stamatatos L. 2004. N-linked glycosylation of the V3 loop and the immunologically silent face of gp120 protects human immunodeficiency virus type 1 SF162 from neutralization by anti-gp120 and anti-gp41 antibodies. J Virol 78:3279–3295. doi:10.1128/JVI.78.7.3279-3295.2004. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
  90. Reynard F, Fatmi A, Verrier B, Bedin F. 2004. HIV-1 acute infection env glycomutants designed from 3D model: effects on processing, antigenicity, and neutralization sensitivity. Virology 324:90–102. doi:10.1016/j.virol.2004.03.022. [PubMed] [CrossRef] [Google Scholar]
  91. Benjouad A, Gluckman JC, Montagnier L, Bahraoui E. 1993. Specificity of antibodies produced against native or desialylated human immunodeficiency virus type 1 recombinant gp160. J Virol 67:1693–1697. [PMC free article] [PubMed] [Google Scholar]
  92. Bolmstedt A, Hinkula J, Rowcliffe E, Biller M, Wahren B, Olofsson S. 2001. Enhanced immunogenicity of a human immunodeficiency virus type 1 env DNA vaccine by manipulating N-glycosylation signals. Effects of elimination of the V3 N306 glycan. Vaccine 20:397–405. [PubMed] [Google Scholar]
  93. Bolmstedt A, Sjolander S, Hansen JE, Akerblom L, Hemming A, Hu SL, Morein B, Olofsson S. 1996. Influence of N-linked glycans in V4-V5 region of human immunodeficiency virus type 1 glycoprotein gp160 on induction of a virus-neutralizing humoral response. J Acquir Immune Defic Syndr Hum Retrovirol 12:213–220. [PubMed] [Google Scholar]
  94. Burke B, Derby NR, Kraft Z, Saunders CJ, Dai C, Llewellyn N, Zharkikh I, Vojtech L, Zhu T, Srivastava IK, Barnett SW, Stamatatos L. 2006. Viral evolution in macaques coinfected with CCR5- and CXCR4-tropic SHIVs in the presence or absence of vaccine-elicited anti-CCR5 SHIV neutralizing antibodies. Virology 355:138–151. doi:10.1016/j.virol.2006.07.026. [PubMed] [CrossRef] [Google Scholar]
  95. Quiñones-Kochs MI, Buonocore L, Rose JK. 2002. Role of N-linked glycans in a human immunodeficiency virus envelope glycoprotein: effects on protein function and the neutralizing antibody response. J Virol 76:4199–4211. doi:10.1128/JVI.76.9.4199-4211.2002. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
  96. McGuire AT, Hoot S, Dreyer AM, Lippy A, Stuart A, Cohen KW, Jardine J, Menis S, Scheid JF, West AP, Schief WR, Stamatatos L. 2013. Engineering HIV envelope protein to activate germline B cell receptors of broadly neutralizing anti-CD4 binding site antibodies. J Exp Med 210:655–663. doi:10.1084/jem.20122824. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
  97. Dowling W, Thompson E, Badger C, Mellquist JL, Garrison AR, Smith JM, Paragas J, Hogan RJ, Schmaljohn C. 2007. Influences of glycosylation on antigenicity, immunogenicity, and protective efficacy of Ebola virus GP DNA vaccines. J Virol 81:1821–1837. doi:10.1128/JVI.02098-06. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
  98. Willett BJ, McMonagle EL, Logan N, Samman A, Hosie MJ. 2008. A single site for N-linked glycosylation in the envelope glycoprotein of feline immunodeficiency virus modulates the virus-receptor interaction. Retrovirology 5:77. doi:10.1186/1742-4690-5-77. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
  99. Cheng-Mayer C, Quiroga M, Tung JW, Dina D, Levy JA. 1990. Viral determinants of human immunodeficiency virus type 1 T-cell or macrophage tropism, cytopathogenicity, and CD4 antigen modulation. J Virol 64:4390–4398. [PMC free article] [PubMed] [Google Scholar]
  100. Pastore C, Ramos A, Mosier DE. 2004. Intrinsic obstacles to human immunodeficiency virus type 1 coreceptor switching. J Virol 78:7565–7574. doi:10.1128/JVI.78.14.7565-7574.2004. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
  101. Reimann KA, Li JT, Voss G, Lekutis C, Tenner-Racz K, Racz P, Lin W, Montefiori DC, Lee-Parritz DE, Lu Y, Collman RG, Sodroski J, Letvin NL. 1996. An env gene derived from a primary human immunodeficiency virus type 1 isolate confers high in vivo replicative capacity to a chimeric simian/human immunodeficiency virus in rhesus monkeys. J Virol 70:3198–3206. [PMC free article] [PubMed] [Google Scholar]
  102. Ratner L, Haseltine W, Patarca R, Livak KJ, Starcich B, Josephs SF, Doran ER, Rafalski JA, Whitehorn EA, Baumeister K, Ivanoff L, Petteway SR Jr, Pearson ML, Lautenberger JA, Papas TS, Ghrayeb J, Chang NT, Gallo RC, 
Wong-Staal F. 1985. Complete nucleotide sequence of the AIDS virus, HTLV-IIINature 313:277–284. [PubMed[]

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