Conformational alterations in the CD4 binding cavity of HIV-1 gp120 influencing gp120-CD4 interactions and fusogenicity of HIV-1 envelopes derived from brain and other tissues.

Abstract

CD4-binding site (CD4bs) alterations in gp120 contribute to HIV-1 envelope (Env) mediated fusogenicity and the ability of gp120 to utilize low levels of cell-surface CD4. In a recent study, we constructed three-dimensional models of gp120 to illustrate CD4bs conformations associated with enhanced fusogenicity and enhanced CD4-usage of a modestly-sized panel of blood-derived HIV-1 Envs (n = 16). These conformations were characterized by a wider aperture of the CD4bs cavity, as constrained by the inner-most atoms at the gp120 V1V2 stem and the V5 loop. Here, we sought to provide further validation of the utility of these models for understanding mechanisms that influence Env function, by characterizing the structure-function relationships of a larger panel of Envs derived from brain and other tissues (n = 81).
Three-dimensional models of gp120 were generated by our recently validated homology modelling protocol. Analysis of predicted CD4bs structures showed correlations between the aperture width of the CD4bs cavity and ability of the Envs to mediate cell-cell fusion, scavenge low-levels of cell-surface CD4, bind directly to soluble CD4, and bind to the Env mAb IgG1b12 whose epitope overlaps the gp120 CD4bs. These structural alterations in the CD4bs cavity were associated with repositioning of the V5 loop.
Using a large, independent panel of Envs, we can confirm the utility of three-dimensional gp120 structural models for illustrating CD4bs alterations that can affect Env function. Furthermore, we now provide new evidence that these CD4bs alterations augment the ability of gp120 to interact with CD4 by increasing the exposure of the CD4bs.

Full text

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Conformational alterations in the CD4 binding
cavity of HIV-1 gp120 influencing gp120-CD4
interactions and fusogenicity of HIV-1 envelopes
derived from brain and other tissues
Gray et al.
Gray et al. Retrovirology 2011, 8:42
http://www.retrovirology.com/content/8/1/42 (2 June 2011)

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SHORT REPORTOpen Access
Conformational alterations in the CD4 binding
cavity of HIV-1 gp120 influencing gp120-CD4
interactions and fusogenicity of HIV-1 envelopes
derived from brain and other tissues
Lachlan Gray1,2, Jasminka Sterjovski1, Paul A Ramsland3,4,5, Melissa J Churchill1,6and Paul R Gorry1,7,8*
Abstract
Background: CD4-binding site (CD4bs) alterations in gp120 contribute to HIV-1 envelope (Env) mediated
fusogenicity and the ability of gp120 to utilize low levels of cell-surface CD4. In a recent study, we constructed
three-dimensional models of gp120 to illustrate CD4bs conformations associated with enhanced fusogenicity and
enhanced CD4-usage of a modestly-sized panel of blood-derived HIV-1 Envs (n = 16). These conformations were
characterized by a wider aperture of the CD4bs cavity, as constrained by the inner-most atoms at the gp120 V1V2
stem and the V5 loop. Here, we sought to provide further validation of the utility of these models for
understanding mechanisms that influence Env function, by characterizing the structure-function relationships of a
larger panel of Envs derived from brain and other tissues (n = 81).
Findings: Three-dimensional models of gp120 were generated by our recently validated homology modelling
protocol. Analysis of predicted CD4bs structures showed correlations between the aperture width of the CD4bs
cavity and ability of the Envs to mediate cell-cell fusion, scavenge low-levels of cell-surface CD4, bind directly to
soluble CD4, and bind to the Env mAb IgG1b12 whose epitope overlaps the gp120 CD4bs. These structural
alterations in the CD4bs cavity were associated with repositioning of the V5 loop.
Conclusions: Using a large, independent panel of Envs, we can confirm the utility of three-dimensional gp120
structural models for illustrating CD4bs alterations that can affect Env function. Furthermore, we now provide new
evidence that these CD4bs alterations augment the ability of gp120 to interact with CD4 by increasing the
exposure of the CD4bs.
Findings
The human immunodeficiency virus type 1 (HIV-1)
envelope glycoproteins (Env) mediate virus entry into
cells and exist as trimers, comprising the surface gp120
glycoproteins noncovalently linked to transmembrane
gp41 glycoproteins that embed the complex into the
viral membrane [1-3]. HIV-1 entry is initiated by gp120
binding to cellular CD4, which facilitates the initial
attachment of virus to the target cell [4]. The binding of
gp120 to CD4 results in dramatic conformational
changes in gp120 that expose the binding site for a
secondary coreceptor, which is either of the chemokine
receptors CCR5 or CXCR4 (reviewed in [5-7]).
Crystallographic and biochemical studies of gp120
have provided valuable insights into mechanisms
involved in CD4 binding and CD4-induced conforma-
tional changes [3,8-12]. The unliganded gp120 core of
simian immunodeficiency virus (SIV) consists of a highly
conserved inner domain which faces the trimer axis and
a heavily glycosylated, globular outer domain which is
mostly exposed on the surface of the trimer [8]. How-
ever, thermodynamic and structural analysis of the
gp120-CD4 interaction demonstrated little evidence of a
structured CD4 binding pocket on the unliganded
gp120, and that CD4bs elements which influence gp120-
CD4 affinity are formed from conformational alterations
* Correspondence: gorry@burnet.edu.au
1Center for Virology, Burnet Institute, Commercial Rd, Melbourne 3004,
Australia
Full list of author information is available at the end of the article
Gray et al. Retrovirology 2011, 8:42
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© 2011 Gray et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in
any medium, provided the original work is properly cited.

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that occur after gp120 has encountered CD4 [2,10]. CD4
interacts with gp120 via surface-exposed residues within
three separate regions distributed over six segments of
gp120. These regions include the a-helices of the inner
domain, the CD4 binding loop of outer domain, and the
b20-b21 ribbon which becomes part of the gp120 brid-
ging sheet, which is a structural element of gp120
formed after CD4 binding that is involved in coreceptor
binding [3,11].
Changes in CD4 binding to gp120 contribute to differ-
ent pathophysiological phenotypes of HIV-1, including
the fusogenic properties of the Env [13,14]. Env med-
iates most of the acute cytopathic effects of HIV-1 infec-
tion in cultured cells [15], and membrane fusion appears
to be an important factor contributing to HIV-1 cyto-
pathicity in vitro [16,17]. In addition, enhancement of
pathogenicity of chimeric simian-HIV (SHIV) strains in
macaques frequently results from increased Env-
mediated fusogenicity [18-22]. Moreover, the cytopathic
effects of Env-mediated HIV-1 fusogenicity are evident
in humans. For example, the presence of multinucleated
giant cells in brain, formed by Env-mediated fusion
between infected and uninfected macrophage lineage
cells, is characteristic of HIV-1 encephalitis and a neuro-
pathological hallmark of HIV-associated dementia [23].
To better understand the molecular mechanisms con-
tributing to alterations in CD4 binding by primary
gp120 proteins and the subsequent influence on Env
function, we recently developed and validated a protocol
to produce and utilize three-dimensional structural
models of gp120 to deduce CD4bs alterations that influ-
ence CD4 binding and Env-mediated fusogenicity [13].
Using a modestly-sized panel of blood derived Envs (n =
16), we showed that a wider aperture of the predicted
CD4bs cavity, as constrained by the inner-most atoms at
the gp120 V1V2 stem and the V5 loop, contributed to
increased fusogenicity and ability of gp120 to bind CD4.
In the present study, we sought to provide further vali-
dation of the utility of these molecular models for
understanding mechanisms that influence Env function,
by characterizing, for the first time, the structure-func-
tion relationships of a larger panel of Envs derived from
brain and other tissues (n = 81).
Production and characterization of a panel of primary
Env clones
Primary HIV-1 viruses isolated from autopsy brain and/
or cerebrospinal fluid, spinal cord, lymph node, spleen
or PBMC from subjects CB1, CB3, MACS1, MACS2,
MACS3, UK1 and UK7 have been described in detail
previously [14,24-27]. The clinical characteristics of the
subjects and coreceptor usage profiles of the primary
viruses are summarized in Table 1. A 2.1 kb fragment
spanning the KpnI to BamHI restriction sites in HIV-1
env (corresponding to nucleotides 6348 to 8478 in
HXB2) was amplified from viral cDNA by PCR and
cloned into the pSVIII-HXB2 Env expression vector
[28], as described previously [29-33]. Between 4 and 6
functional Envs from each virus were identified by entry
assays in JC53 cells with Env-pseudotyped GFP reporter
viruses, as described previously [30,33-35] (Table 1).
The coreceptor specificity of the cloned Envs was deter-
mined by entry assays in Cf2th-CD4/CCR5 and Cf2th-
CD4/CXCR4 cells [35,36] with Env-pseudotyped lucifer-
ase reporter viruses, as described previously [29,35],
which recapitulated the coreceptor usage of the primary
viruses (Table 1). The Envs were sequenced in their
entirety and subjected to multiple sequence alignments
(data not shown) and phylogenetic analysis (Figure 1),
which together showed that the Envs were independent
and compartmentalized according to their tissue of ori-
gin. Thus, we established and characterized a new panel
of Envs (n = 81) derived from autopsy brain and other
tissues of 7 subjects who died from AIDS.
Production of three-dimensional gp120 models and
characterization of the CD4bs cavity
We next produced three-dimensional structural models
of each of the 81 Envs using a protocol that we
described recently [13,32]. Briefly, homology models of
CD4-bound gp120 sequences were prepared using the
Build Model protocol of the Discovery Studio suite, ver-
sion 1.6 (Accelrys, San Diego, CA, USA). This approach
used the Modeller algorithm to generate an atomic
model of the target protein from a template molecule
and a sequence alignment. The template-based models
were optimized by iterative cycles of conjugate-gradient
minimisation against a probability density function that
included spatial restraints derived from the template
and residue specific properties [37]. The crystal struc-
ture of JRFL gp120 containing the V3 variable loop and
bound to CD4 and the X5 Fab antibody fragment was
used as the template for CD4-bound models [9] (Protein
Data Bank ID: 2B4C). The X5 antibody fragment was
deleted from the CD4-bound template prior to model-
ing. The coordinates for gp120 and CD4 were extracted
from the 2B4C crystal structure. Sequence alignments
were generated between JRFL gp120 and the primary
gp120 Env clones. The sequence for CD4 was included
as a second polypeptide chain such that the models of
gp120 were constructed as complexes with CD4. The
V1V2 variable loops were replaced with a GAG linker
sequence and the N- and C- termini overhangs were cut
using the modeling software.
Similarities in three-dimensional structure were mea-
sured by the root mean square deviation (RMSD) of the
distances between main-chain atoms (N, Ca, C and O
atoms) from crystal and model structures after rigid
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body superposition, where an RMSD of < 1Å signifies a
high level of three-dimensional structural similarity
between overlayed proteins. The overall quality of the
geometry of gp120 models generated was verified using
PROCHECK [38].
The three-dimensional structural similarity between
the 2B4C JRFL crystal structure and the 81 predicted
structures of the primary gp120 proteins was < 1.0 Å
for all the primary gp120 models (data not shown), indi-
cating a high overall degree of structural similarity.
Identical RMSD values for each gp120 model were
obtained upon repeated, independent modeling opera-
tions (data not shown). The aperture width of the
CD4bs cavity was deduced from each of the three-
dimensional gp120 structural models by measuring the
distance between the inner most atoms present at the
stem of the V1V2 loops and the V5 loop, which con-
strain the CD4 binding pocket of gp120 [9]. Figure 2
shows the derivation of the CD4bs aperture width for
the Macs2ln5 gp120 model as an example. Analysis of
structural models generated for all the 81 Envs showed
that the aperture width of the predicted CD4bs cavity
ranges from 30 to 36 Å in this panel of Envs.
An increased aperture width of the gp120 CD4bs cavity is
associated with increased fusogenicity, increased CD4-
usage, and increased IgG1b12 Env mAb binding
To determine whether alterations in the width of the
gp120 CD4bs aperture may influence Env function, we
first performed quantitative cell-cell fusion assays with
293T effector cells expressing equivalent levels of Env
on the cell surface, and target cells expressing corecep-
tor and either relatively high or relatively low levels of
CD4 as described previously [13]. In these assays, we
observed positive correlations between the width of the
gp120 CD4bs cavity and the overall level of cell-cell
fusion (Figure 3A), and also with the ability of Env to
utilize low levels of CD4 to mediate cell-cell fusion (Fig-
ure 3B). Next, to better understand the influence of
changes in the gp120 CD4bs aperture on CD4 binding
and CD4bs exposure, we measured the ability of Env to
bind sCD4 and the Env mAb IgG1b12, whose epitope
Table 1 Study subjects, HIV-1 isolates, and summary of Env phenotypes
Subject Risk
factor count
(cells/μl)
Last CD4 Antiretroviral
(s)
HIV-1
encephalitis
Tissues
yielding
HIV-1
isolates
Name of
virus
isolate
Coreceptor
usage of
virus isolate
Envs cloned
from virus
isolate (n)
Functional Coreceptor
usage of
cloned Envs
CB1MH10 ddI (prior
AZT)
SevereBrainCB1-BR X46 YesAll X4
CSF
PBMC
CB1-CSF
CB1-
PBMC
CB3-SC
R5
R5
6
6
Yes
Yes
All R5
All R5
CB3 MH5 ddI (prior AZT
and ddC)
SevereS.Cord R56 YesAll R5
CSF
PBMC
CB3-CSF
CB3-
PBMC
Macs1-BR R5X4
Macs1-
Spln
Macs2-BR R5
Macs2-
LN
Macs3-BR R5
Macs3-
LN
UK1-BR
UK7-BR
R5
R5
6
6
Yes
Yes
All R5
All R5
MACS1MH2 NoneSevereBrain
Spleen
6
6
Yes
Yes
All R5X4
All R5X4R5X4
MACS2MH52AZTModerateBrain
L.Node
5
6
Yes
Yes
All R5
All R5R5
MACS3 MH 95NoneModerateBrain
L.Node
6
6
Yes
Yes
All R5
All R5R5
UK1
UK7
IVDU
IVDU
87
90
ddC (1 mo)
AZT
Moderate
Severe
Brain
Brain
R5
R5
4
6
Yes
Yes
All R5
All R5
The clinical and neuropathological details of the study subjects, and the derivation and characterization of the primary tissue derived HIV-1 isolates have been
published previously [14,24,27], and are summarized again here to assist in the interpretation of the data derived from the cloned Envs. Envs were amplified
from primary virus isolates by PCR and cloned into the pSVIII-Env expression vector as described previously [29,30,33,35]. Functional Envs were identified by
pseudotyping onto Env-deficient GFP reporter virus and entry assays in JC53 cells, as described previously [14,29,30,34,35]. Coreceptor usage of cloned Envs was
determined by pseudotyping onto Env-deficient luciferase reporter virus that were generated in 293T cells, and entry assays in Cf2th-CD4 cells expressing CCR5
or CXCR4, as described previously [30,35]. The coreceptor usage of Envs derived from brain and spleen of subject MACS1 has been reported recently [30]. The
additional Envs described here have been assigned Genbank accession numbers JN001990 to JN002061. Six functional Envs were cloned from Macs2-BR and
UK1-BR viruses, but sequencing and phylogenetic analysis revealed that only 5 and 4 clones, respectively, were independent with unique nucleotide sequences.
Thus, only independent Envs are listed here and included for the subsequent structural and functional analyses. MH, male homosexual; IVDU, intravenous drug
user; mo, month; ddI, didanosine; AZT, zidovudine; ddC, zalcitabine; CSF, cerebrospinal fluid; PBMC, peripheral blood mononuclear cells; S. Cord, spinal cord; L.
Node, lymph node.
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overlaps the gp120 CD4bs, using a subset of Envs (n =
12, Macs1br2-8 and Macs1sp3-15; see Figure 1), as
described previously [13,35]. In these assays, we
observed a near significant association between the
width of the CD4bs cavity and ability of Env expressed
on 293T cells to bind sCD4 (Figure 3C), and a
significant correlation between this parameter and the
ability of Env to bind IgG1b12 (Figure 3D). Together,
these studies, using a large and newly-described panel of
primary Envs, demonstrate the utility of three-dimen-
sional modeling of the gp120 CD4bs cavity for better
understanding the structural basis of Env-CD4
Figure 1 Phylogenetic analysis of env nucleotide sequences. The phylogenetic tree was constructed from an env nucleotide multiple
sequence alignment using a maximum likelihood algorithm, as described previously [39]. The nucleotide sequences of HIV-1 AD8, 89.6, JRCSF,
YU2, NL4-3 and HXB2 env genes were included for comparison. Numbers associated with each branch are bootstrap values obtained from 1000
replicates. Only values above 700 for the major branches are shown. Branch lengths are proportional to the amount of sequence divergence.
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interactions, confirming the results of our recent study
of a different and much smaller panel of Envs [13].
Furthermore, our results provide new evidence suggest-
ing that these predicted CD4bs conformational altera-
tions augment the ability of gp120 to interact with CD4
by increasing the exposure of the CD4bs.
Repositioning of the V5 loop is associated with
conformational alterations in the gp120 CD4bs
To elucidate the gp120 determinants which may contri-
bute to structural alterations in the CD4bs and which
subsequently influence CD4 interactions and fusogeni-
city, we next compared the structural similarity between
CB3sc2 and Macs1br3 gp120 models which are
predicted to have the narrowest and widest of the
CD4bs apertures (30 and 36 Å, respectively). Overlays of
these molecular models revealed a high degree of struc-
tural similarity within the V1V2 stem region, but nota-
ble structural variation within the V5 loop region
(Figure 4A). Furthermore, sequence analysis of all the
primary Env clones showed a relatively high degree of
sequence homology within the V1V2 stem region (Fig-
ure 4B), but a relatively high degree of sequence varia-
tion within the V5 loop (Figure 4C). Together, these
results suggest that, in this new panel of Envs, alteration
in the width of the CD4bs cavity is likely to be due to
sequence variability within the V5 region of gp120
which repositions the V5 loop.
Figure 2 Predicted alterations in the CD4bs cavity from three-dimensional gp120 models. The gp120 model of Macs2ln5 Env is shown
in molecular surface representation, and the CD4 molecule is shown in blue Ca wire, with Phe43 of CD4 highlighted in yellow stick
representation to show the “Phe43 cavity” of gp120. gp120 residues in the CD4 binding pocket located within 4 Å of the CD4 molecule are
shown in ball and stick representation and their molecular surface is highlighted in blue. The width of the CD4bs aperture, as constrained by
the inner-most atoms at the gp120 V1V2 stem and the V5 loop, was deduced as described previously [13].
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Conclusions
Using a large, independent panel of Envs, we confirmed
that structural alterations in the gp120 CD4bs can be
deduced using optimized three-dimensional gp120
molecular models, and that these alterations may influ-
ence fusogenicity and the ability of gp120 to interact
with CD4. We further show, for the first time, that
these alterations appear to increase the exposure of the
Figure 3 The effect of gp120 CD4bs cavity alterations on fusogenicity, CD4-dependence, sCD4 binding and CD4bs exposure. The
CD4bs aperture widths for each gp120 model were plotted against the ability of Env to mediate cell-cell fusion (A), the ability of Env to utilize
low levels of CD4 for cell-cell fusion (B), and the ability of Env to bind sCD4 (C) or the Env mAb IgG1b12 (D), using Prism version 5.0c (GraphPad
Software). The methods for these functional and biochemical assays have been described in detail previously, including the extensive use of
controls to ensure equivalent expression of Env on the cell surface, protocols for generating (and measuring CD4 expression on) CD4lowand
CD4highcells, and the empirical determination of sCD4 and IgG1b12 concentrations used that we showed were within the linear range of Env
binding [13,32,35]. The Spearman correlation coefficient (r) and P values are shown. P values < 0.05 were considered statistically significant. The
data shown are representative of 3 independent experiments.
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CD4bs. Thus, our study provides new insights into
structural mechanisms that contribute to altered interac-
tions between gp120 and CD4. These insights contribute
to a better understanding of HIV-1 entry and in addi-
tion, may inform the design of Env vaccine immunogens
where enhanced exposure of the CD4bs may be desir-
able to elicit effective neutralizing antibody responses.
Furthermore, our modeling approach may be informa-
tive for better understanding structural mechanisms
contributing to HIV-1 disease progression. Here, we
finally describe and characterize a new and relatively
large panel of functional Envs from brain and other
tissues, which will enhance the capacity of investigators
to undertake NeuroAIDS research.
List of abbreviations used
HIV-1: Human immunodeficiency virus type 1; SIV: Simian immunodeficiency
virus; SHIV: Simian-human immunodeficiency virus; Env: HIV-1 envelope
glycoproteins; CD4bs: CD4 binding site; GFP: Green fluorescent protein;
RMSD: Room mean squared deviation; sCD4: soluble CD4; mAb: monoclonal
antibody; BR: Brain; CSF: Cerebrospinal fluid; PBMC: Peripheral blood
mononuclear cells; SC: Spinal cord; Spln: Spleen; LN: Lymph node
Acknowledgements and funding
We thank D. Gabuzda for providing primary HIV-1 isolates, J. Sodroski for
providing pSVIII-HXB2 Env plasmid and Cf2th-CD4/CCR5 cells, J. Sodroski and
Figure 4 Repositioning of the V5 loop is associated with structural alterations in the CD4bs cavity. The gp120 models of CB3sc2 and
Macs1br3 Envs (grey and blue ribbon representation, respectively) were superimposed, and their molecular surfaces were presented as blue or
grey wire mesh (A). The consensus V1V2 stem (B) and V5 loop sequence (C) was deduced for all 81 primary Envs, and the degree of
conservation at each amino acid position was calculated. The GAG linker sequence, which replaced the V1V2 loops in the crystal and model
structures, is shown and highlighted as a grey box in panel (B).
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B. Etemad-Gilbertson for providing Cf2-Luc cells, D. Kabat for providing JC53
cells, and H. Gottlinger for providing pSLV-Tat plasmid.
This study was supported by grants from the Australian National Health and
Medical Research Council (NHMRC) to PRG and MJC (433915 & 433920). LG
is the recipient of a NHMRC Peter Doherty Fellowship. PRG is the recipient
of a NHMRC Level 2 Biomedical Career Development Award. The authors
gratefully acknowledge the contribution to this work of the Victorian
Operational Infrastructure Support Program received by the Burnet Institute.
Author details
1Center for Virology, Burnet Institute, Commercial Rd, Melbourne 3004,
Australia.2Department of Biochemistry and Molecular Biology, Monash
University, Wellington Rd, Clayton 3800, Australia.3Center for Immunology,
Burnet Institute, Commercial Rd, Melbourne 3004, Australia.4Department of
Immunology, Monash University, Commercial Rd, Melbourne 3004, Australia.
5Department of Surgery (Austin Health), University of Melbourne, Studley Rd,
Heidelberg 3084, Australia.6Department of Microbiology, Monash University,
Wellington Rd, Clayton 3800, Australia.7Department of Medicine, Monash
University, Commercial Rd, Melbourne 3004, Australia.8Department of
Microbiology and Immunology, University of Melbourne, Royal Pde, Parkville
3010, Australia.
Authors’ contributions
LG, JS and PRG designed the experiments. LG and JS performed the
experiments. JS and PAR designed the molecular models and interpreted
the modeling data. MJC assisted with Env cloning and sequencing, and
helped interpret the results. PRG supervised the project and helped interpret
the results. LG and PRG wrote the manuscript. All authors helped edit the
manuscript and have read and approved the final version.
Competing interests
The authors declare that they have no competing interests.
Received: 20 April 2011 Accepted: 2 June 2011 Published: 2 June 2011
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doi:10.1186/1742-4690-8-42
Cite this article as: Gray et al.: Conformational alterations in the CD4
binding cavity of HIV-1 gp120 influencing gp120-CD4 interactions and
fusogenicity of HIV-1 envelopes derived from brain and other tissues.
Retrovirology 2011 8:42.
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