Báo cáo khoa học: Retrocyclin RC-101 overcomes cationic mutations on the heptad repeat 2 region of HIV-1 gp41 ppt

Retrocyclin RC-101 overcomes cationic mutations on the
heptad repeat 2 region of HIV-1 gp41
Christopher A. Fuhrman
1
, Andrew D. Warren
1
, Alan J. Waring
2
, Stephen M. Dutz
3
,
Shantanu Sharma
3
, Robert I. Lehrer
2
, Amy L. Cole
1
and Alexander M. Cole
1
1 Molecular Biology & Microbiology, Biomolecular Science Center, Burnett College of Biomedical Sciences at University of Central Florida,
Orlando, FL, USA
2 Department of Medicine, David Geffen School of Medicine, University of California, Los Angeles, CA, USA
3 Department of Chemistry and Center for Macromolecular Modeling & Materials Design, California State Polytechnic University, Pomona,
CA, USA
Defensins are effector molecules of the innate immune
system, which protect humans and other animals from
a wide range of pathogens, including bacteria, fungi
and viruses [1]. There are three major defensin
families (a, b and h). They are classified based on
their b-sheet conformation, cationic charge and
unique disulfide bond pattern [2]. a- and b-defensins

arose from a common pre-mammalian protein [3],
whereas h-defensins evolved directly from a-defensins
[2,4]. h-defensins are formed by the fusion of two
truncated a-defensin nonapeptides by a yet to be iden-
tified mechanism, to form an octadecapeptide that
contains three intramolecular disulfide bonds, and is
macrocyclic through fusion of the N- and C-termini.
Fully translated and processed h-defensins were origi-
nally isolated from the leukocytes of rhesus monkeys,
and intact h-defensin genes exist in Old World
monkeys, orangutans and a lesser ape species [4,5].
Humans, gorillas, bonobos and chimpanzees retain
multiple-mutated, but largely intact h-defensin genes.
Humans express h-defensin mRNA in a variety of
cells and tissues, and their lack of h-defensin peptide
expression is due to a conserved stop codon in the
signal sequence that prevents translation. A search of
the human genome revealed five h-defensin pseudoge-
nes clustered on chromosome 8 near the other a- and
Keywords
AUTODOCK; defensin; HIV-1; innate immunity;
retrocyclin
Correspondence
A. M. Cole, Department. of Molecular
Biology & Microbiology, Burnett School of
Biomedical Sciences, University of Central
Florida, 4000 Central Florida Boulevard,
Building 20, Room 236, Orlando, FL 32816,
USA
Fax: +1 407 823 3635

Tel: +1 407 823 3633
E-mail:
(Received 8 August 2007, revised 24 Octo-
ber 2007, accepted 25 October 2007)
doi:10.1111/j.1742-4658.2007.06165.x
Retrocyclin RC-101, a h-defensin with lectin-like properties, potently inhib-
its infection by many HIV-1 subtypes by binding to the heptad repeat 2
(HR2) region of glycoprotein 41 (gp41) and preventing six-helix bundle for-
mation. In the present study, we used in silico computational exploration
to identify residues of HR2 that interacted with RC-101, and then analyzed
the HIV-1 sequence database at Los Alamos National Laboratory (New
Mexico, USA) for residue variations in the heptad repeat 1 (HR1) and
HR2 segments that could plausibly impart in vivo resistance. Docking
RC-101 to gp41 peptides in silico confirmed its strong preference for HR2
over HR1, and implicated residues crucial for its ability to bind HR2. We
mutagenized these residues in pseudotyped HIV-1 JR.FL reporter viruses,
and subjected them to single-round replication assays in the presence of
1.25–10 lgÆmL
)1
RC-101. Apart from one mutant that was partially resis-
tant to RC-101, the other pseudotyped viruses with single-site cationic
mutations in HR2 manifested absent or impaired infectivity or retained
wild-type susceptibility to RC-101. Overall, these data suggest that most
mutations capable of rendering HIV-1 resistant to RC-101 will also exert
deleterious effects on the ability of HIV-1 to initiate infections – an inter-
esting and novel property for a potential topical microbicide.
Abbreviations
6HB, six helix bundle; gp41, glycoprotein of 41 kDa; HR1, heptad repeat 1; HR2, heptad repeat 2.
FEBS Journal 274 (2007) 6477–6487 ª 2007 The Authors Journal compilation ª 2007 FEBS 6477
b-defensin genes, and one additional h-defensin gene

that had translocated to chromosome 1 [5].
Utilizing genetic information present in the pseudo-
gene, we recreated h-defensins using solid-phase synthe-
sis, and tested for antimicrobial activity [6,7]. The
putative wild-type h-defensin, called retrocyclin or
RC-100, exhibited modest activity against several
Gram-positive and Gram-negative bacteria, yet
potently prevented both X4 and R5 HIV-1 replication
in CD4+ peripheral blood mononuclear cells [6]. A
number of RC-100 analogues have been developed that
are effective in preventing HIV-1 infection, including
the highly active analogue RC-101 [8,9]. RC-101 differs
from ‘wild-type’ retrocyclin-1 by a single arginine-to-
lysine mutation on one of the b-turns. It is nonhemolyt-
ic for human red blood cells, and noncytotoxic against
several human cell lines at concentrations up to
500 lgÆmL
)1
[6,10]. Importantly, RC-101 prevented
infection at low to submicromolar concentrations, and
was active against 27 clinical HIV-1 isolates from five
different clades [8,11].
Retrocyclins act prior to viral entry into host cells by
disrupting the function of glycoprotein 41 (gp41) of
HIV-1 [12,13]. Retrocyclin prevented formation of the
six-helix bundle (6HB) of in vitro synthesized heptad
repeat 1 (HR1) and heptad repeat 2 (HR2) regions of
gp41 [12]. During 100 days of serial passaging, HIV-1
strain BaL developed three cationic mutations in the
presence of sublethal concentrations of RC-101 [14]. Of

the three mutations, one was found in gp120 and one
each in the HR1 and HR2 regions of gp41, and all
three mutations converted a polar or anionic residue to
a cationic residue [14]. In addition, the cationic muta-
tion in HR2 ablated a commonly glycosylated aspara-
gine residue. Loss of glycosylated residues in gp41 can
reduce the fusion ability of the virus, and alter the
shape of discontinuous epitopes [15,16], and thus the
dependence of viral replication on the presence of
RC-101 is not surprising. The cationic mutations and
loss of glycosylated residues suggest an attempt by the
virus to repel the cationic lectin, RC-101 [14,17].
As all nonsynonymous mutations in RC-101-
exposed HIV-1 were cationic mutations [14], and
because HR2 is the principal target of retrocyclins [12],
we decided to study how other mutations that lead to
an increase in net positive charge in HR2 would affect
viral resistance to retrocyclins [18]. Computational
analysis of gp41 revealed a region of low amino acid
diversity in the HR1-binding region of HR2 that was
favored by RC-101 in our docking model. Cationic
mutations revealed only one mutation in HR2 that
offered partial resistance to RC-101; all other muta-
tions showed poor infection, did not infect, or were
inhibited by RC-101 in a manner similar to the wild-
type JR.FL pseudotype.
Results
Variability of amino acids in HR1 and HR2
corresponds to the structural role in the 6HB
conformation

In order to measure the susceptibility of the envelope
gene to mutation and identify viable escape mutants,
we analyzed over 900 HIV-1 group M envelope protein
sequences from the HIV sequence database at Los Ala-
mos National Laboratory (NM, USA). The amino
acid diversity indices of HR1 and HR2 are distinctly
dissimilar (Fig. 1A). The majority of sites (28 of 36)
on HR1 are monomorphic and do not readily change,
whereas the majority of sites (21 of 34) on HR2 are
highly pliable and change readily between viral strains.
Of the eight non-monomorphic sites found on HR1,
six are externally exposed to the environment in the
6HB conformation.
To visualize the sequence variation as a function of
biochemical structure, we mapped the amino acid
diversity values to the 3D model of HR2 (Fig. 1B).
The externally exposed regions of HR2 in the 6HB
show a high amount of amino acid diversity, while the
HR1-binding domain on HR2 is predominantly mono-
morphic. Yamaguchi-Kabata et al. [20] found that dis-
continuous epitopes in the a-helices of gp120 were
under putative positive selection. By contrast, the
monomorphic sites of HR2 suggest a region under
very little selection. Alternatively, the regions exposed
in the 6HB conformation may be under strong puta-
tive positive selection. The long-term potency of
RC-101 against HIV-1 BaL could be attributed to
interaction with discontinuous epitopes of the mono-
morphic residues of HR2.
Because all three known nonsynonymous, RC-101-

evasive mutations were cationic residues [14], we chose
to measure the isoelectric points of the heptad repeat
regions of all group M sequences as a marker of
charge diversity. The isoelectric points of the heptad
repeats illustrate the ability of HIV-1 to alter its regio-
nal charge in vivo. While the amino acid composition
of HR2 is highly variable, its isoelectric range is acidic
and significantly restricted: 96% of the isoelectric
points range between 3.89 and 4.66. In contrast, HR1
is highly monomorphic but covers a wide range of iso-
electric points (Fig. 1C). In line with having only eight
non-monomorphic sites, the isoelectric points show
a strong inclination to cluster around certain values:
8.49 (n ¼ 30), 9.99 (n ¼ 52), 10.29 (n ¼ 33), 10.83
RC-101 overcomes cationic mutations in HIV-1 gp41 C. A. Fuhrman et al.
6478 FEBS Journal 274 (2007) 6477–6487 ª 2007 The Authors Journal compilation ª 2007 FEBS
(n ¼ 569), 11 (n ¼ 140), 11.71 (n ¼ 58) and 12.01
(n ¼ 12). Sequences with higher isoelectric points have
a greater number of cationic mutations with fewer
anionic residues; the converse is true for HR1
sequences with more acidic isoelectric points (data not
shown). The isoelectric range of HR1 is over twice that
of HR2, suggesting a greater in vivo variation in elec-
trostatic density.
The change in free energy upon binding of
RC-101 to HR2 is consistently higher than the
energy of binding to HR1
While charge interaction plays an important role in
RC-101 viral inhibition, it is not known which residues
play an important role in binding. Because RC-101

still binds gp41 in the absence of linked sugar mole-
cules, we can reasonably exclude the sugar moieties
from having a direct interaction with RC-101 [12,14].
The molecular docking program autodock [28,29]
was used to determine the affinity of RC-101 for HR1,
HR2 and the dimer (HR1 + HR2). Previous docking
procedures using the protein models of HR1 and HR2
focused on docking small molecules to the helices [36].
In contrast, RC-101 contains a large number of flexible
side chains and flexible side groups. Consequently, our
docking procedures did not identify just one residue
that can be considered the principal docking site of
RC-101, but a number of RC-101 binding conforma-
tions. Docking of RC-101 to HR1 alone did not result
in a strong binding energy [Fig. 2]. Conversely, the
minimum energy of binding to HR2 is predominantly
lower than values for small molecule inhibitors previ-
ously docked to this model [Fig. 2] [36].
Anionic-to-cationic mutations on HR2 were
unable to elicit appreciable resistance to RC-101
We created HIV-1 env molecular clones to identify
mutations that alter HIV-1 susceptibility to RC-101.
An expression vector containing env from JR.FL,
an R5 pseudotype, was subjected to site-directed
mutagenesis to create mutant clones. Because RC-101
A
B
C
Fig. 1. The ‘a’ and ‘d’ heptamers of HR2 are predominantly mono-
morphic. (A) The amino acid diversity index of HR1 and HR2 was

calculated for 913 group M HIV-1 viruses. All amino acids for which
the index value is below the dotted line (0.05) are considered
monomorphic. (B) The diversity indices were mapped to the 3D
structure of HR2 (N-terminus at the top). Monomorphic residues,
more red in color, are found in the HR1-binding region of HR2.
Highly diverse residues, more white in color, are exposed to the
external environment in the 6HB. (C) Isoelectric points for HR1 and
HR2 were obtained by inputting the group M sequences into the
pI ⁄ M
W
tool of EXPASY. The range of isoelectric points for each axis
has been restricted in order to clearly visualize the isoelectric points
of the majority of HR1 and HR2 molecules.
C. A. Fuhrman et al. RC-101 overcomes cationic mutations in HIV-1 gp41
FEBS Journal 274 (2007) 6477–6487 ª 2007 The Authors Journal compilation ª 2007 FEBS 6479
viral entry inhibition is glycan-independent and charge
alteration is a common mechanism for microbial
evasion of antimicrobial peptides [12,14,37,38], we
individually mutated each negatively charged amino
acid to a positively charged lysine or arginine
(Fig. 3A). After alteration of the env gene, the wild-
type stock (nonmutated) or mutant JR.FL env clones
were then used to create pseudotyped single-cycle
HIV-1 luciferase reporter viruses, and RC-101 activity
against each viral clone was measured. Of the 10
JR.FL variants, five showed scant ability to infect
HOS-CD4-CCR5 cells (Fig. 3B). For all five low- or
noninfectious variants, the mutation was located on
the region of HR2 that is externally exposed in the
6HB conformation (heptamers b, c, e, f and g). Of the

five pseudotyped variants that effectively entered HOS-
CD4-CCR5 cells, only the pseudotype with a lysine at
amino acid position 648 showed partial resistance to
RC-101 (Fig. 3C,D). Residue 648, part of the ‘g’
heptamer, is located in the central region of the helix,
and, based on our modeling simulations, is a potential
binding site for the positive residues on RC-101. These
AB
C
D
Fig. 3. Single-site anionic-to-cationic mutations revealed only one partially resistant variant. The JR.FL env molecular clone was mutated
using site-directed mutagenesis based on the HR2 sequences shown in (A). Pseudotyped viruses were then used to infect HOS-CD4-CCR5
cells. (B) Pseudotypes that infected HOS cells very little or not at all in the absence of RC-101. (C) Pseudotypes that caused infection in a
manner similar to the wild-type JR.FL molecular clone. The percentage inhibition was calculated relative to normal infectious virus. (D) All
the pseudotypes were inhibited similarly to wild-type, except for E648K (P ¼ 0.05). In (A), ‘Hept.’ indicates heptamer location (‘a’–‘g’), as
shown in Fig. 4(C). In (B)–(D), error bars represent the SEM (n ¼ 4).
Fig. 2. RC-101 forms stronger intermolecular bonds with HR2 than
with HR1. Four in silico docking experiments revealed a signifi-
cantly lower DG for RC-101 upon binding HR2 than HR1
(P ¼ 0.0005). The DG upon binding is also referred to as the final
docked energy. Error bars represent the SEM.
RC-101 overcomes cationic mutations in HIV-1 gp41 C. A. Fuhrman et al.
6480 FEBS Journal 274 (2007) 6477–6487 ª 2007 The Authors Journal compilation ª 2007 FEBS
data suggest that the ability of the virus to form the
6HB was significantly decreased and ⁄ or the mutants
lost the ability to properly form the gp41 pre-fusion
complex.
RC-101 binds to the HR1-binding regions of HR2
Figure 4A shows backbone renderings of five RC-101
molecules docked to HR2, representing the five most

energetically favorable dockings in a single docking
simulation. Ligands binding to HR1 were nonspecific,
as evidenced by the highly dispersed RC-101 mole-
cules. In contrast, RC-101 repeatedly bound to HR2
in the same region. Examination of a helical represen-
tation of HR2 (Fig. 4B) shows that the backbone of
RC-101 covers the ‘a’ and ‘d’ heptamers, and the
long flexible side chains of RC-101 extend out and
interact with heptamer locations ‘e’ and ‘g’. Interest-
ingly, these heptamer positions are areas of low
amino acid diversity (Fig. 1B) that coincide with the
region that binds HR1 upon 6HB formation. The
strong affinity of RC-101 for HR2 prevents the inter-
action of HR1 and HR2, formation of the 6HB, and
subsequent fusion of the host and viral membranes
(Fig. 4C), as supported by recent in vitro studies
[12,14].
N & C Terminal
HR2
HR1
Side View
A
B
DE
C
Fig. 4. RC-101 preferentially docks to the HR1-binding domain of HR2. (A) The top five docked RC-101 molecules for a representative dock-
ing, as measured by the final docked energy, are shown as gray loops near the a-helix to which they were docked. The RC-101 mole-
cules docked to HR1 are much more dispersed than the RC-101 molecules docked to HR2. The color of each residue of HR1 and HR2 in (A)
correlates with the heptamer designation shown in (B). (C) RC-101 binds to the HR1-binding region of HR2. An interaction in this region the-
oretically prevents formation of the 6-helix bundle. RC-101 is shown by both (D) cartoon and (E) structural representations.

C. A. Fuhrman et al. RC-101 overcomes cationic mutations in HIV-1 gp41
FEBS Journal 274 (2007) 6477–6487 ª 2007 The Authors Journal compilation ª 2007 FEBS 6481
Anionic, polar and hydrophobic residues of HR2
create a preferred binding site for RC-101
The computer programs ligplot and hbplus were
used to identify specific interactions between the
ligand, RC-101 and HR2 based on proximity and
atomic angles. We quantified the number of interac-
tions per HR2 residue for the lowest (best) 25% of the
docked RC-101 molecules, based on the final docked
energy for each docking experiment comprising 200
iterations of the Lamarckian genetic algorithm
(Fig. 5). This allowed us to isolate regions and residues
of ligand–macromolecule interaction. The applications
identified two sets of molecular interactions between
RC-101 and HR2: hydrogen bonds at residues Ser649,
Gln653 and Asn656, and hydrophobic or nonhydro-
gen-bonded contacts at residues Tyr638, Ile642 and
Leu645 (Fig. 5, asterisks). Five of the six residues are
located in the ‘a’ and ‘d’ heptamer regions of HR2,
which bind HR1 upon 6HB formation. The sixth resi-
due, Gln653, is located on the ‘e’ heptamer. Four of
the residues are monomorphic, and the remaining two
residues have reasonably low amino acid diversity val-
ues. autodock consistently bound RC-101 to a loca-
tion with low amino acid diversity that has an
important role in 6HB formation.
Mutation of residues in the HR1-binding domain
of HR2 resulted in viruses that were not
replication-competent or not resistant to RC-101

Based on the above study, we created mutant pseudo-
typed JR.FL env clones that contained a cationic muta-
tion at each of the six residues observed to interact with
RC-101 in silico. In addition, we mutated two residues
on the 6HB-exposed portion of HR2 (heptamers ‘f’
and ‘c’) as negative controls (Fig. 6A). Both of these
control pseudotypes infected HOS-CD4-CCR5 cells
and remained sensitive to RC-101. Four mutants were
noninfectious even in the absence of RC-101 (Fig. 6B).
All noninfectious JR.FL mutants were located on hep-
tamers that indirectly or directly interacted with HR1
[27,39]. Of the JR.FL mutants that did infect HOS-
CD4-CCR5 cells, none were resistant to RC-101.
Discussion
The envelope protein of HIV-1 is under many kinetic
restraints for proper functionality. First, the short time
between gp120–CD4 interaction and 6HB formation
limits the time that 6HB inhibitors have to act [40,41].
The strong net negative charge of HR2 and net posi-
tive charge of RC-101 create a strong electrostatic
attraction that probably promotes binding. This is evi-
dent in the marked difference observed between the
nonspecific binding of RC-101 to HR1 and the specific
binding to HR2 seen in this work. RC-101 binds
reversibly but with high affinity to glycoproteins and
associates with the cellular lipids and proteins involved
in host–viral fusion [17,42]. This lectin-like binding
places RC-101 in the most advantageous location to
affect 6HB formation.
As a response to opposing host and environmental

factors, HIV-1 employs a number of counter-measures,
including a ‘glycan shield’ and the error-prone nature
of its reverse transcriptase. Alterations in the glycan
Fig. 5. RC-101 dockings prefer both the
polar and hydrophobic residues on HR2.
Four docking experiments were completed,
each involving 200 repetitions of the
Lamarckian genetic algorithm. The best
25% docked RC-101 molecules (n ¼ 50)
from each docking experiment were ana-
lyzed for intermolecular interactions (hydro-
gen bonding and hydrophobic contacts), and
tabulated per HR2 residue. Asterisks indi-
cate the six residues of HR2 that had the
greatest number of interactions with RC-
101, and which were mutated for in vitro
infection assays (Fig. 6). Error bars repre-
sent the SEM.
RC-101 overcomes cationic mutations in HIV-1 gp41 C. A. Fuhrman et al.
6482 FEBS Journal 274 (2007) 6477–6487 ª 2007 The Authors Journal compilation ª 2007 FEBS
shield affect access to binding sites [43]. In addition,
the 6HB formed in solution with the synthetic N36
peptide and a glycosylated C34 peptide was less com-
pact than its nonglycosylated counterpart [16]. This
suggests variation in the interhelical distance, and a
possible change in the discontinuous epitopes targeted
by site-specific antibodies [44]. In our analysis of HIV-
1 protein sequences, we observed many non-monomor-
phic sites with variation primarily within an amino
acid chemical grouping (e.g. Ile M Leu), further alter-

ing possible binding epitopes. The question remains as
to whether HIV-1 mutations that confer partial resis-
tance against RC-101 change the binding site of RC-
101 or alter access to the binding site. Both scenarios
are possible.
In attempting to evade RC-101 inhibition, HIV-1
developed three cat ionic mutations, one of which
removed a glycosylated residue but caused the virus to
remain dependent on RC-101 for infectivity [14]. Anio-
nic-to-cationic mutations in the HR2 region resulted in a
normal infectious mutant only 50% of the time, with all
mutants susceptible to RC-101. This suggests that the
negative charge on HR2 may be important for maintain-
ing the normal replication efficiency of HIV-1, possibly
by stabilizing its interaction with HR1 during 6HB for-
mation. Although mutations that alter the negative
charge of HR2 may impair RC-101 binding, they may
also have the untoward effect (for the virus) of preventing
its ability to mediate the fusion process and infect cells.
The virus’s inability to become fully resistant to RC-
101 is further illustrated by an extension of our previ-
ous work [14]. Passaging the virus from days 100–140
in the presence of 10–20 lgÆmL
)1
RC-101 neither
induced additional mutations nor increased its resis-
tance (data not shown). Collectively, our data indicate
that it is unlikely that HIV-1 can mount further resis-
tance to RC-101: aside from one partially resistant
virus, mutant viruses either remained infectious but

sensitive to RC-101, or suffered from a significant loss
of fusion efficiency.
The predominant problem with current HIV-1 treat-
ments is the eventual emergence of fully resistant
A
B
C
D
Fig. 6. Mutation of residues in the HR1-binding region of HR2 led to noninfectious or non-RC-101-resistant mutants. The JR.FL env mole-
cular clone was mutated using site-directed mutagenesis based on the HR2 sequences shown in (A). Pseudotyped viruses were then used
to infect HOS-CD4-CCR5 cells. Cationic mutations of RC-101-interacting residues revealed nonviable mutations (B) or normally inhibited
mutant pseudotypes (C). (D) All normally infectious mutants were inhibited similarly to the wild-type. In (A), ‘Hept.’ indicates heptamer
location (‘a’–‘g’), as shown in Fig. 4(C). In (B)–(D), error bars represent the SEM (n ¼ 4).
C. A. Fuhrman et al. RC-101 overcomes cationic mutations in HIV-1 gp41
FEBS Journal 274 (2007) 6477–6487 ª 2007 The Authors Journal compilation ª 2007 FEBS 6483
mutants that are then transmitted to new hosts. The
same problem is theoretically possible for widely used
topical microbicides. Our work has shown that the
ability of HIV-1 to generate escape mutants against
RC-101 is limited, and thus RC-101 holds great poten-
tial as an anti-HIV-1 microbicide because it remains
effective against the virus.
Experimental procedures
Computational analysis of the variation
in HR1 and HR2
Aligned envelope protein sequences of 913 unique HIV-1
group M viruses were obtained from the HIV sequence
database at the Los Alamos National Laboratory (http://
www.hiv.lanl.gov), which has been curated by Los Alamos
National Laboratory scientific staff for duplicate sequences

from the same source. HIV-1 group M represents a group
of viral isolates that diverged in humans and originated
from one chimpanzee-to-human transmission event, and
is the most common group found in humans [19].
The amino acid diversity index was calculated as
D
aa
¼
P
20
i¼1
x
i
ÀÁ
2
À
P
20
i¼1
x
2
i
, where x is the proportion of
the ith amino acid of the 20 standard amino acids at that
location [20]. The value is similar to that obtained for gene
diversity in population genetics [21]. An amino acid with a
diversity index less than 0.05 is considered monomorphic
[20]. The residues corresponding to HR1 and HR2 were
spliced out of the sequence file and used for evaluation in
the expasy pI ⁄ M

w
tool to determine the isoelectric point
[22–24].
Preparation of HR1, HR2 and retrocyclin structure
models
Three separate structural representations were required for
bio-computational experimentation: the HR1 and HR2
regions of JR.FL and the h-defensin RC-101. In the context
of computational data, the HR1 ⁄ HR2 nomenclature refers
only to the N36 and C34 peptides, respectively. Three-
dimensional structural models of the HR1 and HR2 regions
of JR.FL were generated using the swiss-model protein
homology web server based on the HIV-1 gp41 core struc-
ture (Protein Data Bank accession number 1AIK) pub-
lished previously [25–27]. The structure for RC-101 was
created using the mutagenesis function of the pymol molec-
ular graphics system, based on the structure of retrocyclin-2
(Protein Data Bank accession number 2ATG). Two in silico
mutations were performed to create RC-101: the second
arginine to a glycine and the fourth arginine to a lysine [9].
The backbone atoms for both mutated residues remained
stationary. There was no need to minimize the rotational
bond energy of the mutated bonds, as all carbon–carbon or
carbon–nitrogen bonds were deemed ‘rotatable’ in the
docking procedure.
Computational modeling of RC-101 binding
‘Grid’ and ‘Docking’ parameter files for all RC-101 doc-
kings to dimer and comparative monomer macromolecules
were prepared using autodocktools (ADT) and accompa-
nying scripts, and then run with autodock 3.0 and auto-

grid 3.0 [28,29]. The grid parameters were the same for all
three macromolecules. The numbers of points in the x, y
and z direction were 76, 76 and 126, respectively. The grid
spacing value was 0.4527 A
˚
. Finally, the grid center was
defined as the x,y,z coordinate (17.449, 13.8, 5.67). All other
autogrid parameters remained at their default values. The
ligand, RC-101, was prepared using ADT according to the
autodock manual [28]. For each macromolecule (HR1,
HR2, HR1 + HR2) and ligand (RC-101), hydrogen posi-
tions were reassigned, nonpolar hydrogens were merged,
and Kollman united charges were assigned to each residue.
The genetic algorithm variables of population size, maxi-
mum number of energy evaluations and the maximum
number of generations were increased to 200, 2 · 10
6
and
2 · 10
5
, respectively, using the methods described by Hete-
nyi et al. [30] as a general guideline. Lower values were
used because the protein model contained substantially less
solvent-exposed surface area and contained less than half
the average number of residues tested in previous blind-
docking studies [30–32]. For each docking simulation, the
genetic algorithm was run 200 times to return 200 possible
RC-101 docked conformations. autodock reports the
change in free energy upon binding for each conformation.
An approximate threshold range (– 9 to )11 kcalÆmol

)1
)
separates nonspecific interactions from prominent inter-
molecular bonds. Each docking simulation was executed
four times, and the quantitative measures of all four dock-
ing simulations were averaged and the SEM calculated.
Defining hydrogen and nonhydrogen bonds
Tables of hydrogen bonds and nonhydrogen bonds were
generated using ligplot in conjunction with hbplus
[33,34]. The best 25% of the docked RC-101 molecules
(n ¼50), according to the final docked energy, were tabu-
lated, and the mean number of bonds per residue for four
independent docking executions was reported by the pro-
gram, together with the SEM.
Preparation of peptide
The 18-amino-acid peptide RC-101 was synthesized as pre-
viously described [4,6,35] with the sequence: cyclic-GIC-
RCICGKGICRCICGR. After each step, the peptide was
subjected to MALDI-TOF mass spectrometry to assess
RC-101 overcomes cationic mutations in HIV-1 gp41 C. A. Fuhrman et al.
6484 FEBS Journal 274 (2007) 6477–6487 ª 2007 The Authors Journal compilation ª 2007 FEBS
homogeneity (typically approximately 95%), and to confirm
that the observed mass agreed with the theoretical mass.
Peptide concentrations were determined by quantitative
peptide analysis.
Cell culture
HOS-CD4-CCR5 cells (N. R. Landau, Salk Institute for
Biological Studies, La Jolla, CA, USA), which allow entry
of R5 HIV-1, were acquired from the National Institutes of
Health AIDS Research and Reference Reagent Program

(Germantown, MD, USA). HOS cells were grown in
DMEM supplemented with penicillin, streptomycin, 10%
fetal bovine serum, 1 lgÆmL
)1
puromycin, and mycophenol-
ic acid selection medium. 293T cells were grown in DMEM
with penicillin, streptomycin and 10% fetal bovine serum.
HIV-1 plasmid constructs and viral entry assay
The expression vectors pNL-LucR
–
E
–
and JR.FL env were
gifts from N. R. Landau. JR.FL is an R5 strain of HIV-1. In
total, 18 JR.FL env mutants were constructed. Nine glutamic
acids on the HR2 of HIV-1 gp41 were mutated to lysines.
Amino acids 632, 634, 641, 647, 648, 654, 657 and 659 were
mutated from Glu (GAA) to Lys (AAA). Amino acid 636
was mutated from Asp (GAC) to Arg (CGC). For the second
set of mutagenesis studies, polar or hydrophobic residues
were mutated to an arginine or lysine: Tyr638 (TAC) to Arg
(CGC), Ser640 (AGC) to Arg (AGG), Ile642 (ATA) to Lys
(AAA), Leu645 (CTA) to Arg (CGA), Ser649 (TCG) to
Lys (CGC), Asn651 (AAC) to Lys (AAA), Gln653 (CAA)
to Lys (AAA), and Asn656 (AAT) to Lys (AAA).
Each mutation was created from the wild-type JR.FL env
plasmid using the QuikChange multi site-directed mutagen-
esis kit (Stratagene, La Jolla, CA, USA), verified by
sequencing (University of Central Florida Biomolecular
Science Center Genomics Core Laboratory, Orlando, FL,

USA) and compared to the published JR.FL wild-type
sequence (accession number U63632). Subsequently, HIV-1
single-cycle (replication-incompetent) luciferase reporter
viruses were produced by cotransfecting 293T cells with
10 lg each of pNL-LucR
–
E
–
and one of the JR.FL env
clones. Virus-containing clarified supernatants were col-
lected after 48 h by centrifugation at 1000 g for 10 min,
filtered though a 0.45 lm filter and stored at )80 °Cin
aliquots until needed. One aliquot was used to quantify
propagated pseudovirus by p24 ELISA (Perkin Elmer, Wal-
tham, MA, USA). Another aliquot was used to ensure the
integrity of the envelope gene. Viral RNA was isolated
from the JR.FL pseudotypes (viral RNA mini kit; Qiagen,
Valencia, CA, USA). A cDNA library was created from
the isolated viral RNA using the iScript Select cDNA syn-
thesis kit (Bio-Rad, Hercules, CA, USA). Then a 666 bp
envelope region containing HR1 and HR2 was PCR-ampli-
fied, and the DNA was separated using a 1.5% agarose gel.
The sense primer used was 5¢-CTGTGTTCCTTGGG
TTCTTGG-3¢, and the antisense primer was 5¢-CTCCACC
TTCTTCTTCGATTCC-3¢. To measure the infectious abil-
ity of the JR.FL pseudotypes, HOS-CD4-CCR5 cells
(5 · 10
3
per well; 96-well plate) were infected with 50 ng
p24 per well of virus in the presence or the absence of

RC-101 (0, 1.25, 2.5, 5 or 10 lgÆmL
)1
), and luciferase
activity was measured 2 days later.
Acknowledgements
This work was supported by grants from the National
Institutes of Health: AI052017, AI065430 and
AI060753 (to AMC) and AI056921 (to RIL), and from
the National Science Foundation: EIA-0321333 (to
SS). We thank Martin Kline (UCF) for his excellent
technical help and Dr G. M. Morris (The Scipps
Research Institute) for autodock support. We are
also grateful to the National Biomedical Computation
Resource and Dr W. Li (NBCR) for the use of their
computer grid cluster. SS thanks Professor
P. W. Mobley (California State Polytechnic University)
for insightful discussions of the computational results,
and CAF thanks Dr C. Parkinson (UCF) for helpful
discussions on molecular evolution.
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