Primary HIV-1 R5 isolates from end-stage disease display enhanced viral fitness in parallel with increased gp120 net charge.

Abstract

To better understand the evolution of the viral envelope glycoproteins (Env) in HIV-1 infected individuals who progress to AIDS maintaining an exclusive CCR5-using (R5) virus population, we cloned and sequenced the env gene of longitudinally obtained primary isolates. A shift in the electrostatic potential towards an increased net positive charge was revealed in gp120 of end-stage viruses. Residues with increased positive charge were primarily localized in the gp120 variable regions, with the exception of the V3 loop. Molecular modeling indicated that the modifications clustered on the gp120 surface. Furthermore, correlations between increased Env net charge and lowered CD4(+) T cell counts, enhanced viral fitness, reduced sensitivity to entry inhibitors and augmented cell attachment were disclosed. In summary, this study suggests that R5 HIV-1 variants with increased gp120 net charge emerge in an opportunistic manner during severe immunodeficiency. Thus, we here propose a new mechanism by which HIV-1 may gain fitness.

Full text

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Primary HIV-1 R5 isolates from end-stage disease display enhanced viral fitness in
parallel with increased gp120 net charge
Johanna Repitsa, Jasminka Sterjovskib,c, Daniel Badia-Martinezd, Mattias Milda,e,i, Lachlan Grayb,f,
Melissa J. Churchillb,c, Damian F.J. Purcellf, Anders Karlssong, Jan Alberth,i, Eva Maria Fenyöa,
Adnane Achourd, Paul R. Gorryb,c,f,⁎,1, Marianne Janssona,h,i,⁎,1
aDepartment of Laboratory Medicine, Lund University, Sweden
bMacfarlane Burnet Institute for Medical Research and Public Health, Melbourne, Australia
cDepartment of Medicine, Monash University, Melbourne, Australia
dCenter for Infectious Medicine, Karolinska Institutet, Karolinska University Hospital Huddinge, Stockholm, Sweden
eDepartment of Experimental Medical Science, Lund University, Sweden
fDepartment of Microbiology and Immunology, University of Melbourne, Australia
gDepartment of Infectious Medicine, South Hospital, Stockholm, Sweden
hDepartment of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Stockholm, Sweden
iDepartment of Virology, Swedish Institute for Infectious Disease Control, Solna, Sweden
a b s t r a c ta r t i c l e i n f o
Article history:
Received 11 April 2008
Returned to author for revision
28 April 2008
Accepted 11 June 2008
Available online 30 July 2008
Keywords:
HIV-1
R5
AIDS
Env
gp120
Net charge
Evolution
Viral fitness
To better understand the evolution of the viral envelope glycoproteins (Env) in HIV-1 infected individuals
who progress to AIDS maintaining an exclusive CCR5-using (R5) virus population, we cloned and sequenced
the env gene of longitudinally obtained primary isolates. A shift in the electrostatic potential towards an
increased net positive charge was revealed in gp120 of end-stage viruses. Residues with increased positive
charge were primarily localized in the gp120 variable regions, with the exception of the V3 loop. Molecular
modeling indicated that the modifications clustered on the gp120 surface. Furthermore, correlations
between increased Env net charge and lowered CD4+Tcell counts, enhanced viral fitness, reduced sensitivity
to entry inhibitors and augmented cell attachment were disclosed. In summary, this study suggests that R5
HIV-1 variants with increased gp120 net charge emerge in an opportunistic manner during severe
immunodeficiency. Thus, we here propose a new mechanism by which HIV-1 may gain fitness.
© 2008 Elsevier Inc. All rights reserved.
Introduction
The reverse transcriptase enzyme of human immunodeficiency
virus type 1 (HIV-1) has a low fidelity and lacks proof-reading
capacity. This results in a high mutation rate of approximately one
point mutation per genome per replication cycle (Mansky and Temin,
1995). Consequently, within an infected individual viral quasispecies
of distinct but closely related viruses are established. With 1010new
virions produced every day in combination with the high mutation
rate, the quasispecies population is very variable (Perelson et al.,
1996). In constant interplay with selective forces of the host and
therapeutic agents, the quasispecies population evolves continuously
during the course of infection (van Opijnen and Berkhout, 2005). Thus,
the intra host virus evolution can be traced by analyzing the changes
in the viral genome over time.
The viral envelope gp120/gp41 trimeric complex (Env), which is
physically exposed to the host immune system has been shown to
exhibit the greatest genetic diversity among the HIV-derived proteins
(Hahn et al., 1985). Previous studies have revealed correlations
between variations in the gp120 sequence and the function of the
virus. Mutations in the variable loops (V1, V2 and V3) of gp120,
resulting in length variations and charge differences, are related to
altered cell tropism (Hoffman and Doms, 1999).
Virology 379 (2008) 125–134
⁎^Corresponding authors. P.R. Gorry is to be contacted at Macfarlane Burnet Institute
for Medical Research and Public Health, GPO Box 2284, Melbourne 3001, Victoria,
Australia. Fax: +61 3 9282 2100. M. Jansson, Swedish Institute for Infectious Disease
Control, Nobelsvag 18, 171 82 Solna, Sweden. Fax: +46 8337460.
E-mail addresses: johanna.repits@med.lu.se (J. Repits), jasminka@burnet.edu.au
(J. Sterjovski), Daniel.Badia-Martinez@ki.se (D. Badia-Martinez),
Mattias.Mild@med.lu.se (M. Mild), lachlang@burnet.edu.au (L. Gray),
churchil@burnet.edu.au (M.J. Churchill), dfjp@unimelb.edu.au (D.F.J. Purcell),
anders.karlsson@sodersjukhuset.se (A. Karlsson), jan.albert@smi.se (J. Albert),
Eva_Maria.Fenyo@med.lu.se (E.M. Fenyö), Adnane.Achour@ki.se (A. Achour),
gorry@burnet.edu.au (P.R. Gorry), marianne.jansson@smi.se (M. Jansson).
1These authors contributed equally to this work.
0042-6822/$ – see front matter © 2008 Elsevier Inc. All rights reserved.
doi:10.1016/j.virol.2008.06.014
Contents lists available at ScienceDirect
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To gain entry into target cells HIV-1 binds to CD4 via the viral
glycoproteingp120 which initiates a seriesof events includingbinding
of a coreceptor, CCR5 and/or CXCR4, and ultimately gp41-mediated
fusion of the viral and cell membranes (Pierson and Doms, 2003).
CCR5-restricted (R5) viruses predominate in early, asymptomatic
stages of infection (van't Wout et al., 1994). Emergence of viruses
able to use CXCR4 instead of- or in addition to CCR5 for cell entry has
been correlated to rapid progression to AIDS (Bjorndal et al., 1997).
However, approximately 50% of infected individuals progress to AIDS
while maintainingexclusive use of CCR5 (de Roda Husman et al.,1999;
Jansson et al., 1999; Karlsson et al., 1994; Koot et al., 1993). We and
others have previously described late stage R5 HIV-1 variants, isolated
from AIDS patients, with altered biological properties compared to
viruses isolated before AIDS onset (Gorryet al., 2005; Grayet al., 2005;
Janssonetal.,1999;Janssonetal.,1996;Karlssonetal.,2004;Koninget
al., 2003; Kwa et al., 2003; Repits et al., 2005; Sterjovski et al., 2007).
Our previous studies on sequential R5 isolates obtained before and
after AIDS onset revealed an evolution of the R5 phenotype with
respect to enhanced viral fitness, altered mode of coreceptor use and
reduced sensitivity to inhibition by CCR5 ligands and HIV-1 entry
inhibitors (Gray et al., 2005; Jansson et al., 1999; Jansson et al., 1996;
Karlsson et al., 2004; Repits et al., 2005; Sterjovski et al., 2007).
In the present study we sought to better understand the evolution
of the HIV-1 R5 envelope of primary isolates during disease pro-
gression. Multiple full-length gp160 env sequences from R5 isolates,
obtained before and after AIDS onset, were analyzed. We demonstrate
the emergence of R5 variants with gp120 molecules that display an
increased positive net charge at the end-stage HIV-1 infection. Muta-
tions leading to an altered amino acid charge were mapped mainly to
the variable regions but excluding the V3 loop. Structural modeling
suggested that the modified residues were clustering and localized on
the gp120 outer surface. Results also revealed that end-stage viruses
displayed enhanced ability to associate with cells independently of
CD4andCCR5expression.Thenetchargeofgp120alsocorrelatedwith
the CD4 count at time of virus isolation, virus attachment ability,
replicative capacity, enhanced infectivity and reduced sensitivity to
several entry inhibitors. Thus, after AIDS onset these features may
evolve, in an opportunistic manner, and result in the emergence of
HIV-1 R5 variants with enhanced pathogenic properties.
Results
Analysis of sequentially obtained R5 env sequences
To examine the molecular evolution of the HIV-1 R5 envelope
glycoproteins during progressive disease, from the chronic phase to
end-stage disease, we analyzed 48 env sequences (GenBank:
EF600067–EF600114). These sequences were obtained from R5
primary isolates sequentially obtained before and after AIDS onset
in six patients (Table 1). To determine the evolutionary relationships
between the selected env sequences, maximum likelihood phyloge-
netic trees were constructed. Sequences from each patient clustered
Fig. 1. Evolutionary relationships of env sequences encoding gp160 obtained from
chronic and end-stage R5 viruses. Maximum likelihood trees were constructed using
PAUP⁎ (Sinauer Associates, Inc Publishers) with heuristic searches. Statistical support of
the trees was obtained by 100 bootstrap replicates using the LUNARC computer cluster
at Lunds University [http://www.lunarc.lu.se].
Table 1
Patient clinical status, CD4 count, time to/from AIDS diagnosis and virus coreceptor use
Patienta
IsolateCD4bcountMonthscto AIDS
−9
+26
−27
+6
−30
+11
−11
+20
−54
+20
−2
+16
Clinical statusCoreceptorduse
G 1228
4481
624
3899
5013
8616
1372
5714
668
7363
6322
8004
260
5
290
6
140
90
220
20
750
20
200
9
Chronic asympt.
End-stage AIDS
Chronic asympt.
End-stage AIDS
Chronic asympt.
End-stage AIDS
Chronic asympt.
End-stage AIDS
Chronic asympt.
End-stage AIDS
Chronic asympt.
End-stage AIDS
CCR5
CCR5
CCR5
CCR5
CCR5
CCR5
CCR5
CCR5
CCR5
CCR5
H
I
J
M
R CCR3+CCR5
CCR3+CCR5
aPatient code according to Jansson et al. (1999).
bCD4+T cells/μl at time of virus isolation.
cTime point of virus isolation related to months before and after AIDS diagnosis.
dCoreceptor use determined by infection of U87.CD4 and GHOST (3) coreceptor
indicator cell lines expressing CCR2b, CCR3, CCR5, CXCR4, CXCR6 or BOB (Jansson et al.,
1999).
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together and were separated from the sequences from all other
patients by a bootstrap value of 100%. In addition, sequences from
each isolation time point formed separate sub-clusters (Fig. 1). This
phylogenetic analysis excludes contamination between patient speci-
mens and/or isolation time points. In further analysis we examined
the complete gp160 (Env) sequence as well as shorter segments, i.e.
gp120, gp41 and gp120 variable loop regions.
Env of end-stage R5 viruses display increased net charge
The development of CXCR4-using viruses has been correlated to
increased positive charge in specific Env regions (Hoffman and Doms,
1999). In the light of this we analyzed alterations in the charge of R5
Env sequences from primary isolates obtained before and after AIDS
onset. We found that the gp160 sequences from end-stage disease of
all patients displayed higher positive net charge when compared to
corresponding sequences obtained prior to AIDS onset (Fig. 2a,
p=0.028, Wilcoxon's matched pairs test). Mutations leading to an
increase in positive charge were mapped to gp120, while no such
pattern was observed for gp41 (Figs. 2b and c, p=0.028 and not
significant respectively, Wilcoxon's matched pairs test). The exact
location of amino acids with increased charge varied between the
patients. However, we noted that mutations resulting in increased net
charge mainly were localized in the variable gp120 regions (Fig. 2d),
except the V3 loop, which was highly conserved when comparing Env
sequences from chronic and end-stage R5 viruses. Nevertheless, three
out of the six patients had end-stage R5 viruses with an increased
charge in the regions flanking the gp120 V3 loop (Fig. 2d). In addition,
it should be noted that among the gp120 V-regions an increased
positive net charge was most frequent within the V2 and V4 regions
since end-stage R5 Env sequences in a majority of the patients
displayed increased positive charge within these regions (Fig. 2d and
data not shown). To investigate the structural localization of the
increased positive net charge within gp120 of end-stage R5 viruses
we created molecular models of gp120 based on the Env sequences
obtained before and after AIDS onset from patient G. The molecular
models were created using the program SWISS-MODEL (Arnold et al.,
2006), based on the homology of our Env sequences to the previously
published crystal structure of HIV-1 gp120 (Huang et al., 2005). The
molecular models comprise the core as well as the V3, V4 and V5
loops, but the V1 and V2 loops are excluded (Fig. 3a). As illustrated in
Figs. 3b and c the molecular models indicate that, besides the
observed increase in basicity within the V2 loop, most of the
positively charged residues that were introduced following AIDS
onset clustered on the surface of the gp120 structure, including the V4
loop, forming a novel basic region. It should be noted that the models
suggest that most of the residues composing this relatively positively
charged region protrude towards the solvent. Furthermore, most of
this novel basic region is localized on the V4 loop, well separated from
the previously described basic domain localized at the base of gp120
(that faces the target cell) (Kwong et al., 2000; Moulard et al., 2000) as
well as the CD4 binding region (Wyatt and Sodroski, 1998) (Figs. 3b
and c). It is also important to note that our model underestimates the
overall gp120 charge differences since the observed positive shift in
the V1V2 region net charge (Fig. 2d) is not displayed in the model.
Accordingly, we conclude that gp120 of R5 virus variants emerging
after AIDS onset undergoes substitutions that result in an increased
net positive charge. These changes shift the amino acid net charge of
gp120 and cluster to the V2 and V4 loops on an exposed region of the
gp120 outer surface.
Fig. 2. Evolution and localization of charge modifications of HIV-1 R5 Envamino acid sequences during end-stage disease progression. Differences in net charge of a) gp160, b) gp120
and c) gp41 comparing the average charge of four R5 sequences per isolate obtained longitudinally at the asymptomatic chronic phase and after AIDS onset. d) Schematic view of
localization of amino acid charge modifications within gp160 calculated from the average of four sequences per R5 isolate. Illustrated are amino acid positions where increased (filled
triangles) or decreased (open triangles) positive charge have been noted comparing the end-stage Env sequences with corresponding chronic phase Env sequences.
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Increase in gp120 net positive charge correlates with patient immune
status at time of R5 virus isolation
We next investigated whether the observed evolution in net
charge was related to the immune status of the patients (Table 1). We
compared the Env net charge with the CD4 count at time of virus
isolation. The correlation analyses showed a stronginverse correlation
between the CD4 count and the net charge of gp160 as well as gp120
(Figs. 4a and b) (p=0.00005, R=−0.90 and p=0.0006, R=−0.84,
respectively according to Spearman rank correlation). In contrast,
gp41 net charge did not correlate with CD4 count at time of virus
isolation (Fig. 4c). These results suggest that invivo evolution of the R5
HIV-1 gp120 during progressive disease may result in significant
molecular changes such as increased positive net charge, which
correlate with the immune status of the patient.
gp120 net charge correlates with properties related to viral fitness
We previously reported that R5 viruses that emerged after AIDS
onset showed enhanced fitness, including elevated replicative
capacity, increased infectivity and reduced sensitivity to several
entry inhibitors (Gray et al., 2005; Jansson et al.,1996, 1999; Karlsson
Fig. 3. Molecular models of monomeric and trimeric gp120 derived from sequentially obtained chronic and end-stage R5 viruses from patient G. a) Molecular surface representation
of a model of monomeric gp120. A Cα tracing of the two N-terminal domains of CD4 is colored inyellow. The variable loops V1V2 (stem), V3, V4 and V5 are colored in blue, magenta,
green and red, respectively. The rest of the surface of the molecule is colored grey. b) Electrostatic surfaces of chronic (left) versus end-stage (right) gp120. The electrostatic potential
is shown at the solvent-accessible surface, which is colored according to the local electrostatic potential, ranging from dark blue (most positive) to deep red (most negative).
c) Trimeric models of chronic (left) versus end-stage (right) gp120 derived from R5 Env clones from patient G. The trimer is depicted from the orientation of the viral membrane.
Fig. 4. Correlations between CD4 count at time of virus isolation and net charge of Envsequences. CD4 count in correlation to a) gp160, b) gp120 and c) gp41 net charge. Presented net
charges are the average of four Env sequences per R5 isolate.
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et al., 2004; Repits et al., 2005; Sterjovski et al., 2007). To examine the
underlying nature of these biological changes we investigated the
relationship between molecular alterations leading to increased
positive net charge in gp120 and markers of viral fitness. The analysis
revealed clear correlations between the gp120 net charge and viral
replicative capacity in PBMC, infectivity in PBMC and infectivity in
U87.CD4.CCR5 (p=0.0004, p=0.005 and p=0.008 respectively accord-
ing toSpearman rank correlation)(Figs.5a–c). Thenet charge of gp120
also correlated withR5 virus sensitivity to inhibitionbythree different
entry inhibitors, the fusion inhibitor T-20, the CCR5 antagonist TAK-
779, and the natural CCR5 ligand RANTES (p=0.042, p=0.0005 and
p=0.0007 respectively according to Spearman rank correlation)
(Figs. 5d–f). Taken together, these results imply that increased viral
fitness is correlated to the net charge of gp120.
End-stage R5 virus displays enhanced attachment to both CD4+CCR5+
and CD4−CCR5−cells
Next, we investigated if the observed molecular changes i.e.
increased positive net charge at the outer surface of the gp120 crown,
affect the ability of viruses to associate with target cells. We
performed virus attachment assays where virus was allowed to attach
to cells, that either expressed specific receptors, i.e. CD4 and CCR5, or
not. The amount of cell bound virus was calculated as the % p24
antigen, out of added p24, that remained after incubation, washing
and cell lysis. Our results showed that end-stage viruses had an
increased ability to attach to CD4+CCR5+cells when compared to the
corresponding chronic viruses as assessed using two different cell
lines (Fig. 6a) (p=0.004; Wilcoxon's matched pairs test). End-stage
virusesalsodisplayed enhancedattachmenttoCD4andCCR5negative
cells (Fig. 6b) (p=0.005; Wilcoxon's matched pairs test). Furthermore,
the enhanced cell attachment of end-stage viruses was noted both
when virus inoculum was normalized to the same level of functional
RT(Figs.6a andb) as wellas thep24 antigencontent(datanot shown).
In addition, cell-attachment ability correlated to the net charge of
gp160 (Figs. 6c and d). This was true for cells expressing CD4 and CCR5
as well as receptor negative cells (p=0.002, R=0.81 and p=0.0005,
R=0.85 respectively, Spearman rank correlation). Similarly, the
gp120 net charge tended to correlate with degree of R5 virus
attachment to both receptor positive and negative cells (Figs. 6e and f)
(p=0.060, R=0.56 and p=0.077, R=0.53 respectively, Spearman rank
correlation).Thus,theresultsindicatethatR5variantswithanenhanced
ability to attach to cells, independent of specific receptor expression,
may evolve at end-stage of disease, and this ability correlates with Env
amino acid net charge.
Discussion
By analysis of multiple Env sequences from sequentially obtained
HIV-1 R5 isolates this study reveals molecular rationales for in vivo
evolution of the R5 phenotype during severe immunodeficiency. In
our studies of patients who maintain HIV-1 viruses exclusively using
CCR5 we show that virus variants expressing gp120 with increased
positive net charge emerge at end-stage disease along with declining
CD4+T cell counts. The mutations leading to increased positive net
charge of gp120 were mapped to the surface of the protein and mainly
to the V2 and V4 variable regions. We also demonstrate correlations
between gp120 net charge and different parameters related to the
fitness of HIV-1 R5 isolates.
Both our work and that of others have demonstrated an in vivo
evolution of the R5 phenotype during disease progression in patients
thatmaintainCCR5 restricted HIV-1viruses(Gorryet al.,2005; Grayet
al., 2005; Jansson et al.,1996,1999; Karlsson et al., 2004; Koning et al.,
2003; Kwa et al., 2003; Repits et al., 2005; Sterjovski et al., 2007). R5
virus variants with altered biological properties, such as increased
resistance to inhibition by CCR5 ligands and other entry inhibitors,
may evolve during disease progression (Gray et al., 2005; Jansson et
al., 1996, 1999; Karlsson et al., 2004; Koning et al., 2003; Kwa et al.,
2003; Sterjovski et al., 2007; Sterjovski et al., 2006). The R5 variants
emerging during disease progression also display enhanced cyto-
pathicity and increased infectivity along with altered quantitative and
qualitative demands on target cell receptors and enhanced sensitivity
to neutralizing antibodies (Grayet al., 2005; Karlsson et al., 2004; Kwa
et al., 2003; Repits et al., 2005; Sterjovski et al., 2007).
Our analysis of Env sequences from longitudinally obtained
isolates revealed that gp120 from R5 isolates obtained at the end-
stage of the disease display a higher net charge compared to viruses
isolated at the chronic stage of HIV-1 disease. The mutations leading
to increased charge were predominantly located in the variable
regions of gp120, and most commonly in the V2 and V4 regions. In
Fig. 5. gp120 net charge in relation to properties of viral fitness. Correlations between the gp120 net charge and a) replicative capacity in PBMC as assessed by p24 release,
b) infectivity evaluated as TCID50 in PBMC, c) infectivity evaluated as plaque-forming units in U87.CD4.CCR5 cells, d) sensitivity to inhibition by fusion inhibitor T-20, e) sensitivity to
inhibition by TAK-779 and f) sensitivity to inhibition by the natural CCR5 ligand RANTES.
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contrast, the charge of the V3 loop, which in many previous studies
has been shown to be an important region for receptor interactions
(Fouchier et al., 1992; Hwang et al., 1991), was conserved between all
paired chronic and end-stage R5 env sequences. These findings
suggest that changes in the net charge of the V3 loop may not be
favored by CCR5-restricted viruses within patients that maintain
exclusive R5 virus populations throughout the entire disease course.
This is also in accordance with studies demonstrating that an
increased V3 charge is indicative of a shift from CCR5- to CXCR4-use
(Fouchier et al., 1992; Hoffman and Doms, 1999; Hwang et al., 1991).
The variable regions of gp120 are flexible loops protruding from
the main body of the Env trimer, and are involved in receptor
binding (Huang et al., 2005; Kwong et al., 1998). It is therefore
reasonable to assume that the variable regions of gp120 are
important in the initial contact between the virus and the target
cell. Since the surface of the target cell as well as the overall virus
surface are negatively charged the electrostatic repulsion must be
overcome for successful virus binding. Comparing the V3-loop
charge of non-syncytium inducing and syncytium inducing viruses
it was previously hypothesized that increased positive charge of
gp120 resulted in an initial enhanced interaction between virus- and
target cell membranes, as a result of reduced repulsion (Callahan,
1994). Also, a recently proposed physical model of the initial steps of
retrovirus infection suggests that the initial force for virus adsorption
Fig. 6. Attachment of chronic and end-stage R5 viruses to CD4+CCR5+and CD4−CCR5−cells. Results are presented as % p24 of chronic and end-stage R5 viruses bound to a) CD4+CCR5+
cells; black diamonds = Cf2.CD4.CCR5 and gray triangles = NP-2.CD4.CCR5, or b) CD4−CCR5−cells; black lines = 293T and grey lines = NP-2wt. Correlations between average % p24
bound to c) and e) CD4+CCR5+cells or d) and f) CD4−CCR5−cells and c) and d) gp160 and e) and f) gp120 net charge.
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is provided by non-specific electrostatic interactions (Davis et al.,
2004). To counteract the electrostatic repulsion between membranes
and aid virus adsorption a cationic polymer such as polybrene might
be added to in vitro cultures (Davis et al., 2002). We previously
reported that R5 isolates from end-stage disease do not benefit from
such cationic assistance upon infection of PBMC (Repits et al., 2005).
An overall increased positive net charge of the outermost parts of
gp120 might provide these viruses an increased ability for non-
specific adherence to the cell surface by reduced electrostatic
repulsion between the viral and cell membranes. This may also
explain why R5 viruses emerging at end-stage disease are less
dependent on cationic assistance for target cell binding and entry.
Further support for the evolution of R5 viruses with enhanced
adherence capacity can be derived from our observation that end-
stage R5 viruses have an increased ability to attach to cells when
compared to corresponding isolates obtained prior to AIDS onset.
Interestingly, end-stage viruses had an elevated ability to attach to
cells as compared to chronic-stage viruses, which was independent of
receptor expression. This ability was furthermore shown to correlate
tothe net charge of gp160. Interestingly, the correlation betweenvirus
cell-attachment ability and net positive charge was stronger for gp160
than for gp120. Thus, it is possible that the charge of certain amino
acid residues within gp41 may contribute to this viral property, even
though cell-attachment ability was not correlated with the net charge
of gp41 when analyzed separately (data not shown). Hence, these
end-stage R5 viruses exhibit an unspecific adherence trait, which
could be explained by the increased positive charge of the Env surface.
Enhanced Env net charge may also account for our recent report on
more promiscuous use of the CCR5 receptor by end-stage R5 viruses,
as assessed by broadened chimeric receptoruse (Karlsson et al., 2004).
In a parallel study we noted that R5 virus variants emerging
following AIDS onset also display reduced numbers of potential N-
linked glycosylation sites (PNGS) in the V2 and V4 regions (Borggren
et al., 2008). Since glycans not only are bulky, but also negatively
charged (Le Doux et al., 1996) loss of glycans may additionally
contribute tothe elevation of the overall Envcharge and thus facilitate
and enhance virus–cell interactions. Furthermore, loss of glycans may
reduce solubilityof the glycoproteins, and onepotential mechanism to
compensate for the reduced solubility might be to increase the net
charge, and the hydrophilic properties, of the gp120 backbone. Thus,
the observed increased basicity within the gp120 V2 and V4 regions
may facilitate loss of glycans, which in turn might result in a more
openpre-triggered Env structure. Interestingly, this positivelycharged
region does not correspond to the CD4-binding region (Wyatt et al.,
1998). Furthermore, this basic region was localized away from the
previously described conserved basic region of the gp120 trimer that
faces away from the virus, towards the target cell membrane (Kwong
et al., 2000; Moulard et al., 2000).
Even if the molecular models suggest that the novel basic region
within gp120 does not overlap with the CD4 binding site, a shift in the
electrostatic potential that enlarge the basic surface of gp120 along
with loss of glycans may render these viruses more fusogenic. A basic
surface that does not comprise the CD4 binding site, nor the V3 loop,
but conserved parts of the coreceptor binding site within gp120 have
been identified and implicated in tropism and sensitivity of HIV-1 to
polyanions (Moulard et al., 2000). Thus, we believe that the impact of
altered gp120 charge and glycosylation patterns for HIV-1 entry
mechanisms merits further investigations.
We also demonstrate a clear inverse correlation between the
gp120 net charge and the immune status of the patient, as assessed by
numbers of CD4+T cells at time of virus isolation. Further support for
Env changes during the progressive loss of immunological compe-
tencewas derived fromcalculations of dN/dS ratios. Both in gp120 and
in gp41 these dN/dS ratios were found to be lower than 1.0, (data not
shown) suggesting a comparatively low overall selection pressure for
amino acid change in Env during progressive disease, which would be
in agreement with previous studies (Williamson et al., 2005).
However, despite the fact that the average positive Darwinian
selection pressure for amino acid change was low, we clearly showed
that amino acids in specific gp120 regions evolved towards higher
charge. We believe that these changes may evolve as a result of
diminished immune pressure. Thus, our data suggest that decreased
strength of the host immunity may serve as a selection force at end-
stage HIV infection,akintothe selectiontowards consensussequences
that has been reported in CTL epitopes when HIV is released from
HLA-specific targeting during HIV transmission (Leslie et al., 2004). It
is tempting to draw parallels between R5 viruses at the end-stage of
the disease and those evolving in the environment of a primary HIV-1
infection. Hence, even though increased fitness may not be the result
of the same viral properties, we believe that the selection mechanisms
resulting in enhanced fitness could be comparable in the acute- and
end-stages of the disease when a potent immune response is lacking.
However, it is possible that reduced availability of target cells and
altered receptor density also could contribute to the selection of
viruses with increased fitness and altered Env structures (van Opijnen
and Berkhout, 2005).
In the present study we reportthatalong with decliningCD4+Tcell
counts, mutations in gp120 result in increased positive charge of the
protein surface and that these sequence changes correlate well with
the viral fitness of the R5 viruses fromwhich theyoriginate. In support
of this observation we have in a different set of R5 isolates, cross-
sectionally obtained, also noted higher gp120 net charge in R5 isolates
obtained after AIDS onset (Gorry and Sterjovski, unpublished data).
We believe that the enlarged basic surface of gp120 in end-stage R5
HIV-1 may enhance non-specific electrostatic interactions between
viral and cell membranes and aid adsorption of these viruses onto
target cells. Thus, we propose a new mechanism that HIV-1 may use
for the gain of viral fitness, which in turn could contribute to the
pathogenicity of end-stage HIV-1 R5 viruses.
Materials and methods
Patients and virus isolates
Studied HIV-1 isolates were obtained from six patients selected
from a larger cohort of homo- and bisexual men attending the South
Hospital, Stockholm, Sweden (Table 1) (Karlsson et al.,1991; Karlsson
et al., 1994). The selection was made on the basis of our previous
finding that in patients that maintain R5 viruses throughout the
course of the disease, R5 viruses with enhanced fitness and reduced
sensitivity to RANTES and entry inhibitors may appear with progres-
sion to AIDS (Jansson et al., 1996, 1999; Repits et al., 2005). Four of
these patients (G, I, J and R) received antiretroviral monotherapy (AZT
or ddI). Isolations were made sequentially, at the chronic stage when
the patients were clinically asymptomatic and after progression to
AIDS. Virus stocks were generated by passaging virus isolates in PHA-
stimulated (Boule) peripheral blood mononuclear cells (PBMC) from
healthy donors. The R5 phenotype was determined by infection of the
coreceptor indicator cell lines GHOST and U87 (Jansson et al., 1999).
Isolates from patient R (6322 and 8004, see Table 1) displayed the
ability to use both CCR5 and CCR3 in the indicator cell lines. However,
since these isolates did not replicate in PBMC carrying the homo-
zygous CCR5 Δ32 genotype (Jansson et al., 1999), they were classified
being of R5 phenotype.
Generation of full-length env clones
Genomic DNA was extracted from PBMC infected with the HIV-1
R5 isolates seven days post infection, using a DNeasy DNA extraction
kit (Qiagen) according to the manufacturer's protocol. HIV-1 envgenes
were amplified from genomic DNA using Expand high fidelity DNA
polymerase and nested PCR approach as described previously (Gray et
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al., 2006; Ohagen et al., 2003). The outer primers were Env1A and
Env1M (Gao et al., 1996) and the inner primers were Env-KpnI and
Env-BamHI (He et al.,1997) whichamplifies a 2.1 kbfragmentof HIV-1
env corresponding to nucleotides 6348 to 8478 of HxB2 and spans
unique KpnI and BamHI restriction sites. PCR was performed with an
initial denaturation step of 94 °C for 2 min, followed by 29 cycles of
95 °C for 15 s, 60 °C for 30 s, and 72 °C for 2 min, and a final extension
of 72 °C for 7 min. During the last 20 cycles the extension time was
increased byan additional 5 s per cycle. PCR product DNAwas purified
over a column using High Pure PCR Product Purification Kit (Roche)
and cloned into the pSVIIIenv expression plasmid (Gao et al., 1996;
Ohagen et al., 2003) by replacement of the 2.1 kb KpnI to BamHI HxB2
env fragment. Thus, the cloned env fragments contain the entire gp160
coding region except for 36 amino acids at the N-terminus and 105
amino acids at the C-terminus, which in the pSVIII plasmid derived
from HxB2.
Sequence analysis of env clones
The pSVIIIenv plasmid was used as template for sequence analysis
of the env gene and from each R5 isolate four clones were selected
according to functionality in a single round entry assay described
previously (Gorry et al., 2005; Gray et al., 2006). A set of 7 forward;
F1EnvJR (5′-G/CAGAAAGAGCAGAAGACAGTGGCAATGA-3′), F2EnvJR
(5′-GTCTATTATGGGGTACCTGTGTGG-3′), F3EnvJR (5′-GTGTACCCA-
CAGACCCCAACCCACAAG-3′), F4EnvJR (5′-ACAATGC/TACACATGGAAT-
TAA/GGCCA-3¢), F5EnvJR (5′-TTTAATTGTGGAGGGGAATTTTTCT-3′),
F6EnvJR (5′-GTGGGAATAGGAGCTATGTTCCTTGGG-3′), F7EnvJR
(5′-TATCAAAC/TTGGCTGTGGTATATAA-3′) and 8 reverse primers;
R1EnvJR (5′-CTATCTGTCCCCTCAGCTACTGCTA-3′), R2EnvJR (5′-GCT-
AAGAATCCATCCACT-AATCGT-3′), R3EnvJR (5′-CCTGCCTAACTCTATT-
CAC-3′), R4EnvJR (5′-TTCAATTAG/AGGTGTATATTAAGCCTGTG-3′),
R5EnvJR (5′-GCCCCAGACTGTGAGTTGCA-ACAGATG-3′), R6EnvJR (5′-
GATGGGAGGGGCATACAT-3′), R7EnvJR (5′-CAGCAGTTGAGTT-
GATACTACTGG-3′), R8EnvJR (5′-TTTAGCATCTGATGCACAAAATAG-3′)
spanning the entire gp160 region and the ABI prism BigDye
Terminator sequencing kit (Perkin Elmer) were used in the sequen-
cing reaction. Sequence analysis was performed at the SWEGENE
Centre of Genomic Ecology at Lund University. The sequenced
segments were assembled to a contig sequence using the ContigEx-
press of VectorNTI Advance 10 software (Invitrogen). Sequences were
aligned using ClustalX (Xia and Xie, 2001) followed by manual editing
in GeneDoc [http://www.psc.edu/biomed/genedoc]. In order to rule
out contamination between specimens a maximum likelihood tree
containing sequences from all clones was constructed. The best-
fitting nucleotide substitution model was identified with Modeltest
(Posada and Crandall, 1998). Maximum likelihood trees were
constructed using PAUP⁎ (Sinauer Associates, Inc Publishers) with
heuristic searches. Statistical support of the trees was obtained by 100
bootstrap replicates using the LUNARC computer cluster at Lunds
University [http://www.lunarc.lu.se]. The gp160 sequences were
analyzed as full-length segments and shorter regions i.e. gp120,
gp41, gp120 variable regions. We defined the variable regions of
gp120 as follows; V1 (nucleotide 6615–6692 in the HxB2 sequence),
V2 (6693–6812), V3 loop (7110–7217), V4 (7377–7478) and V5 (7596–
7637). The net charge of the sequences was calculated with each
lysine (K) and arginine (R) contributing +1 and each aspartic acid (D)
and glutamine (E) contributing −1. All presented charges were
calculated as the average of four sequences per isolate. The charge
difference at specific positions within gp160 was compared between
end stage and chronic stage AIDS R5 isolates and ranged accordingly
from −2 (four clones with basic aa replaced by four clones with acidic
aa) to +2 (four clones with acidic aa replaced by four clones with basic
aa). Phylogenetic analysis was performed using MEGA version 3.1
(Kumar et al., 2001). Pairwise nucleotide distances were computed
using MEGA 3.1 under the Tamura–Nei nucleotide substitution model
with gamma distributed rates among sites (gamma parameter α=0.5)
and pairwise deletions.
Molecular modeling of chronic- and end-stage gp120
The molecular models of the gp120 monomers werecreated on the
basis of their sequence homology to previously solved crystal
structures of gp120, using the SWISS-MODEL protein modeling server
(Guex and Peitsch, 1997). The crystal structure of gp120 in complex
with CD4 and the X5 antibody ((Huang et al., 2005); pdb code 2B4C)
was used as templatefor the creation of preliminary models of chronic
and end-stage gp120. Fragments of the gp120 models, mainly from
loop domains, were rebuilt according to secondary structure align-
ment. The molecular models of the trimeric gp120 were based on the
previously published model of a trimeric gp120 (Posada and Crandall,
1998), kindly provided by Dr Peter D. Kwong and Dr Marie Pancera.
The models of monomeric gp120 were superposed on each of the
components of the trimer. The coordinates of all models will be
provided upon request.
Virus cell-attachment assay
The ability of the studied viruses to attach to cells expressing the
specific receptors, CD4 and CCR5, as well as unspecific binding to
cells not expressing these receptors was tested. For this purpose we
used NP-2 and Cf2th cells expressing CD4 and CCR5, in addition to
NP-2wt and 293T cells being negative for CD4 and CCR5 expression.
All cells were cultured in DMEM supplemented with 10% FCS (vol/
vol), 0.1 μg/ml Streptomycin and 0.1 U/ml Penicillin. For selection of
CD4 and CCR5 in NP-2 cells, 500 μg/ml Neomycin and 1 μg/ml
Puromycin were added. For selection of CD4 and CCR5 in Cf2th/CD4
+CCR5+cells 0.5 mg/ml G418 and 0.1 mg/ml hygromycin were added.
Cells were seeded 1 day prior to the addition of virus. The
concentration of functional RT has been shown to be a more
accurate measurement of infectious virions compared to the p24
content (Corrigan et al., 1998; Malmsten et al., 2003; Marozsan et al.,
2004). To avoid bias we tested in parallel the inoculum virus
normalized to the same level of functional RT (2 or 5 ng/ml) and p24
content (10 ng/ml).150 μl virus was allowed to attach for 3 h at 37 °C
and there after the cells were extensively washed in PBS and
resuspended in 100 μl lysis buffer (0.5% [vol/vol] NP-40; 0.5% [wt/
vol] sodium deoxycholate; 50 mM NaCl; 25 mM Tris–HCl [pH 8.0];
10 mM EDTA, 5 mM benzamidine HCl; and a cocktail of protease
inhibitors). The level of virus attachment was analyzed by calculating
% p24 antigen, out of added p24, that remained after incubation,
washing and cell lysis using a p24 antigen ELISA (BioMérieux)
according to the manufacturers' instructions.
Virus infectivity and replication assays
The infectivity and the replicative capacity of the studied HIV-1 R5
isolates were analyzed as described (Repits et al., 2005). In brief,
infectivity was analyzed by determination of 50% tissue culture
infectious dose (TCID50) in donor PBMC and plaque-forming units
(PFU)/ml in U87.CD4.CCR5 cells in inoculum virus normalized for
functional reverse tranciptase (RT) activity. Replicative capacity was
evaluated by the analysis of p24 antigen release in cultures of donor
PBMC infected with RT normalized R5 isolates.
Entry inhibitor sensitivity assay
The sensitivity of the HIV-1 R5 isolates to entry inhibitors was
analyzed as described (Repits et al., 2005). In brief, donor PBMC were
infected by R5 viruses in the presence of dilutions of CCR5-ligand
RANTES, small molecule CCR5 antagonist TAK-779, and fusion
inhibitor T-20. Sensitivity to the entry inhibitors was then evaluated
132
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Page 9

as 50% inhibitory concentrations calculated from the release of p24
antigen from control cultures infected in the absence of inhibitors.
Statistical analysis
For statistical analysis we used the Statistica software version 7.
The non-parametric Spearman rank correlation was used for the
analysis of correlations, while at comparisons between chronic and
end-stage Env sequences and virus properties Wilcoxon's matched
pairs test was used.
Acknowledgments
We thank Dr Joseph Sodroski for providing the pSVIIIenv
expression plasmid and Cf2th-CD4/CCR5 cells, Dr Hiroo Hoshino for
the NP-2 cell lines, Dr Daniel Littman for U87.CD4.CCR5 cell line, Dr
Peter D Kwong and Dr Marie Pancera for molecular models and
Hannes Uchtenhagen for suggestions on the manuscript. T20 fusion
inhibitor from Roche and TAK-779 were obtained through the NIH
AIDS Research and Reference Reagent Program, Division of AIDS,
NIAD, NIH. DNA sequencing was performed at the SWEGENE Centre of
Genomic Ecology at Lund University supported by the Knut and Alice
Wallenberg Foundation through the SWEGENE consortium. The work
was supported by grants provided to MJ from the Swedish Research
Council and to MJ and EMF from the Swedish International
Development Agency/Department for Research Cooperation (Sida/
SAREC). Grants were also provided by the Magn. Bergvall's Founda-
tion, the Physicians Against AIDS Research Foundation, Clas
Groschinskys Foundation, the Royal Physiographic Society in Lund,
Sweden. JR was given a travel grant from the Solander Foundation for
a six month visit to the laboratory of PRG. PRG was supported in part
by a grant from the Australian National Health and Medical Research
Council (NHMRC) (433915). JS and LG were supported by Australian
NHMRC Dora Lush Biomedical Postgraduate Research Scholarships.
PRG is the recipient of an Australian NHMCR R. Douglas Wright
Biomedical Career Development Award.
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