HIV Fusion Peptide Perturbs Membrane Structure in a Cholesterol Dependent Fashion


90a Sunday, February 3, 2013
is currently unknown. In this work, we use density gradient centrifugation and
liposome coflotation analysis to demonstrate that the anchoring energy of the
Dengue E protein in anionic membranes composed of POPCþPOPG is much
greater than in membranes composed of POPCþPOPE.

469-Pos Board B238
HIV Fusion Peptide Perturbs Membrane Structure in a Cholesterol
Dependent Fashion
Alex Liqi Lai, Jack H. Freed.
ACERT, Cornell University, Ithaca, NY, USA.
The fusion between viral envelopes with host cell membranes is required for
viral entry and infection, which is mediated by special glycoproteins anchored
on the viral membrane. Fusion peptide (FP) is the domain that initiates mem-
brane fusion. However, the mechanism of membrane fusion is still unclear.
We previously found that the influenza hemagglutinin FP increases the order
of DMPC lipid bilayer. We hypothesize that inducing lipid ordering might
be a critical step in fusion caused by a variety of fusion proteins. HIV gp41
FP plays a similar role as influenza HA FP. However, gp41 FP is polymorphic
and changes from alpha helix to beta aggregation as cholesterol concentration
in lipid increases. We used PC spin labels on the lipid head group and different
positions on the acyl chain to detect the perturbation by gp41 FP to POPC/
POPG lipid bilayers with different cholesterol concentration (0% to 30%) by
CW-ESR. Our data show that 1) gp41 FP affects the lipid order in the same pat-
tern as HA FP does, i.e., a cooperative effect vs. lipid/peptide ratio, thus sup-
porting our hypothesis; 2) gp41 FP induces membrane ordering in all tested
lipid compositions, consistent with promoting membrane fusion in these com-
positions; 3) in the high cholesterol containing lipid bilayers, whereas gp41 FP
is in the beta aggregation conformation, its effect on the lipid ordering reaches
deeper into the bilayer, consistent with deeper membrane insertion for gp41 FP
in this conformation. We are extending the ESR studies to look for coexisting
membrane microdomains induced by different conformations of FPs and the FP
partitioning between them and for precise separation of the effects of ordering
and molecular motion.

470-Pos Board B239
CD4 Binding Induces an Asymmetric Transition of HIV-1 Env from its
Native Conformation into the Prehairpin Intermediate State
Mukta D. Khasnis, Konstantine Halkidis, Anshul Bhardwaj, Michael Root.
Thomas Jefferson University, Philadelphia, PA, USA.
HIV-1 membrane fusion is mediated by sequential binding of the viral homo-
trimeric Env (gp120/gp41) to cellular CD4 and chemokine receptor (CXCR4 or
CCR5). These binding events coordinate conformational transitions of the
gp41 trimer from its native conformation into a prehairpin intermediate state
(PHI) and, subsequently, into its fusogenic trimer-of-hairpins structure
(TOH). In the PHI, the gp41 trimer assumes an extended conformation that
bridges the viral and cellular membranes, and exposes the N-HR and C-HR do-
mains which are targeted by HIV-1 fusion inhibitors T20 and 5-Helix, respec-
tively. Our previous work suggests that the PHI-to-TOH transition occurs in
a concerted (symmetric) fashion. Here, we investigate the transition from the
native state to the PHI using a functional complementation strategy that em-
ployed heterotrimeric Envs containing only one or two functional CD4- and
chemokine receptor-binding sites. Additionally, by incorporating T20- and
5-Helix- resistance mutations into individual Env protomers, we were able to
interrogate exposure of discrete gp41 N-HR and C-HR domains. Our data
indicate that a single CD4 binding to an Env trimer is sufficient to promote
fusion. Moreover, the first CD4 binding event appeared to expose only one
of three possible binding sites for each fusion inhibitor, suggesting an
asymmetric transition into the PHI. This asymmetry was significantly more
pronounced in the gp41 N-HR region compared to the gp41 C-HR region.
These results can be used to explain the multiphasic titration of mutant Env
homotrimers that naturally form asymmetric N-HR domain structures. Taken
together, these data suggest that the native state-to-PHI transition of Env occurs
in a multistep (asymmetric) fashion.

471-Pos Board B240
Evolution of HIV-1 Resistance to a Cholesterol-Linked D-Peptide Fusion
Inhibitor
Amanda E. Siglin1, Nicholas Francis2, Michael S. Kay2, Micheal J. Root1.
1
Thomas Jefferson University, Philadelphia, PA, USA,

2
University of Utah,

Salt Lake City, UT, USA.
During HIV-1 entry, the N-HR and C-HR regions of the gp41 viral glycoprotein
associate to form a trimer-of-hairpins critical for membrane fusion. The N-HR
region contains a highly conserved hydrophobic pocket that is the target for
a multivalent, D-peptide fusion inhibitor, PIE12-trimer. Recently, a modified
form of this inhibitor with a conjugated cholesterol (chol-PIE12-trimer) was
shown to possess up to 160-fold increased potency, presumably due to mem-
brane targeting. Here, we determined the resistance profile of chol-PIE12-
trimer starting from either inhibitor-naı̈ve or PIE12-trimer-resistant HIV-1
(NL4-3 strain). Viral propagation in increasing inhibitor concentrations pro-
duced HIV-1 populations with 50- to 100-fold reduced sensitivity chol-
PIE12-trimer. These viruses were also resistant to the parental, unconjugated
inhibitor PIE12-trimer. Using HIV-1 fusion inhibitors di-C37 and 5-Helix,
we were able to interrogate the effect of escape mutations on the exposure of
the gp41 N-HR and C-HR regions, respectively. Resistance to chol-PIE12-
trimer resulted in a slight decrease in the temporal window of N-HR exposure,
regardless of the starting viral population. Exposure of the C-HR region was
unchanged for resistant viruses generated from the inhibitor-naı̈ve viral pool.
By contrast, the temporal window of C-HR exposure was significantly
increased (>20-fold) for resistant viruses that were generated from the
PIE12-trimer-resistant viral pool. Efforts to identify the mechanisms of chol-
PIE12-trimer resistance and the decoupling of N-HR and C-HR exposure are
underway.

472-Pos Board B241
Fusogenic Activity of the HIV-1 Gp41 MPER-TMD Region: Mechanism
and Targeting by Immunogens and Inhibitors
Jose L. Nieva1, Beatriz Apellaniz1, Carmen Domene2, Nerea Huarte1,
Eneko Largo1.
1
Biophysics Unit (CSIC-UPV/EHU) and Dept. of Biochemistry, University
of the Basque Country (UPV/EHU), Bilbao, Spain,

2
Chemistry Research

Laboratory, University of Oxford, Oxford, United Kingdom.
Fusion of the viral envelope with the cell membrane marks the beginning of
the HIV-1 replicative cycle. This event is targeted by inhibitors currently in
clinical use and by preventive vaccines under development. Antibodies 4E10
and 10E8 bind to the gp41 membrane proximal external region (MPER)-
transmembrane domain (TMD) junction and block fusion. These antibodies
display the broadest viral neutralization known to date, which underscores
the conservation and functionality of the MPER-TMD region. In recent
work, we have described that peptides representing this region have potent
membrane-destabilizing effects. Here, based on the outcome of vesicle
assays, atomic force microscopy studies and molecular dynamics simulations,
we propose a mechanism for the involvement of the MPER-TMD region
in HIV-1 fusion. In addition, we provide evidence that underpins the poten-
tial use of its activity as a new target for inhibitor and immunogen
development.

473-Pos Board B242
Studies on the Membrane-Active Behavior of Outer Membrane Vesicles
from a Gram Negative Bacterium
William Bartos, Rensa Chen, Donald Y. Kobayashi, Paul R. Meers.
Rutgers, The State University of New Jersey, New Brunswick, NJ, USA.
Gram-negative bacteria are known to produce small ~50-200 nm vesicles by
‘‘blebbing’’ of their outer membranes. These outer membrane vesicles
(OMV) have been implicated in activities such as transmission of virulence fac-
tors, horizontal gene transfer and development of biofilms. In this investigation,
the interactions of OMV from Lysobacter enzymogenes (strain C3) with other
membranes have been monitored using fluorescent assays for association
and/or fusion. Defined composition large unilamellar vesicles labeled with
the fluorescence resonance energy transfer (FRET) pair 1,2-dipalmitoyl-sn-
glycero-3-phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl) (NBD-
PE) and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rho-
damine B sulfonyl) (Rh-PE) were incubated with isolated OMV to observe the
interaction via the FRET ratio of donor/acceptor. OMV substantially increased
this ratio over the course of about an hour (t1/2 ~10-20 min.) at 30

�C, when
added directly to vesicles comprising disordered or fluid lipids, such as
1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1’-rac-glycerol) (POPG), 1-pal-
mitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) or 1-palmitoyl-2-
oleoyl-sn-glycero-3-phosphocholine (POPC). This apparent fusion was much
more limited or absent with liquid ordered membranes of 1,2-dipalmitoyl-sn-
glycero-3-phosphocholine/cholesterol (DPPC/chol). In the case of POPG/
POPE 1/3 vesicles, apparent fusion was also confirmed by testing vesicles
with exclusively inner monolayer fluorescent probes to diminish any fluores-
cence changes from external interactions of outer monolayer fluorophores.
When the OMV were labeled with the fluorophore 1,10-dioctadecyl-3,3,30,30-
tetramethylindocarbocyanine, and incubated with a natural host, yeast cells
(strain S30), association and/or fusion was observed by co-sedimentation of
the OMV label with yeast cells. These studies may help to elucidate the mech-
anism of natural host-pathogen interactions and define the lipid specificity for


	HIV Fusion Peptide Perturbs Membrane Structure in a Cholesterol Dependent Fashion
	CD4 Binding Induces an Asymmetric Transition of HIV-1 Env from its Native Conformation into the Prehairpin Intermediate State
	Evolution of HIV-1 Resistance to a Cholesterol-Linked D-Peptide Fusion Inhibitor
	Fusogenic Activity of the HIV-1 Gp41 MPER-TMD Region: Mechanism and Targeting by Immunogens and Inhibitors
	Studies on the Membrane-Active Behavior of Outer Membrane Vesicles from a Gram Negative Bacterium