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V3 Loop Binding to CCR5 and CXCR4 of Rapid and Non Progressor HIV Patients in
Baltimore MD
Background
The HIV virus is a growing epidemic in the world that
is responsible for the cause of death of millions of
individuals. Modern medicine has not has found a
cure and the high mutations that occur make the
virus even harder to suppress once inside the body.
The HIV virus is made of to key domains: the inner
core and viral membrane. The purpose of this
poster was to follow point mutations that occur in the
V3 loop on the gp120 and CD4 counts of 2 needle
using patients in Baltimore MD who have contracted
the HIV virus. A non-progressor, the CD4 count did
not drop throughout the study, and a rapidprogressor, the CD4 count dropped drastically
through the study, patients V3 loop of the gp120 on
the viral membrane were studied to find a reason as
to why one patient died from HIV and the other did
not die.
The V3 loop binds to CCR5 and CXCR4 receptors
found on the T-cell in the body. Research has
shown that HIV that can utilize the V3 loop to bind
CCR5 and CXCR4 is the most lethal combination for
the destruction of the host T-cells. The V3 loop
binds to the receptors through specific and
nonspecific binding techniques and allows for the
virus to enter into the T-cell leading to death of that
T-cell. In this study it was possible to see as to why
the rapid-progressor did indeed die from the actions
of the HIV virus where as the non-progressor lived
with a high CD4 count. These reasons all relate
back to point mutations along the V3 loop and the
structural alignment between the V3 loop when
interacting with the CCR5 and CXCR4 receptors.
V3 loop of gp120
Figure 1: Ribbon Diagram of V3 Loop; On the left is the conformation of the V3 loop in
water. The Right picture show the conformation of the V3 loop in a polar solvent
mimicking the actual binding conformation.
The V3 loop is found on the gp120 protein and has a specific 3
dimensional structure that facilitates the HIV virus to binding to CCR5 and
CXCR4 receptors on T-cells. There are two different sequences of the
V3 loop: R5 and X4. For the purposes of this poster the R5 will only be
considered. The V3 loop consists of 36 amino acids and has 2 stem and
1 crown domain as seen in the sequences bellow. The V3 loop begins
with a disulfide bond between C1 and C36. The N-terminal stem consist
of a conserved N-terminal loop and a small section of the β strand. The
C-terminal stem is a conserved loose α helix. The crown region consists
of a variable β strand, a conserved β hairpin turn, and a variable ridged
loop. The β hairpin turn contains a highly conserved GPG sequence that
is seen in all HIV isomers. The stem regions are believed to bind to the
N-terminal domains of the CCR5 and CXCR4 receptors through salt
bridges and nonspecific binding. The crown region of the V3 loop binds
more specifically to the ECL2 of CCR5 and CXCR4 receptors utilizing the
variable regions surrounding the β hairpin turn motif. Research has
shown that the variable regions can undergo rapid point mutations that
favor the binding of CCR5 or CXCR4. The ribbon diagram above show
the conformation change that the V3 loop undergoes when it is in a water
solution (Left) to a more polar solution (Right) that is typically seen upon
binding to the different receptors. The correct 3 dimensional structure is
necessary for aligning the crown region to the ECL2 allowing for viral
binding.
Rapid-progressor
Patient 10
Patient 10 was considered to be a rapid-progressor in
the study done in Baltimore MD. This patient appears to
have survived to the end of the study although with an
extremely low CD4 count of 15 on visit 6. Looking at the
CD4 count of this patient between visit 2 and visit 3
there was a significant drop in the patients CD4 count.
The overall sequences of visits 1 and 2 were combined
as well as the sequences from visits 3 and 4 which all
showed similar V3 sequences. Comparing the
sequences from visit 1&2 to the sequence from 3&4
there are point mutations that occur that shift the binding
affinity from the CCR5 receptor the favor binding to the
CXCR4 receptor. Using the point mutation study,
substation of amino acids R11, R18, F20, and R30
showed that these amino acids affected the binding to
CXCR4 but not the binding to CCR5. The amino acids
that are conserved between visits 1-4 were R30 and
R18. Therefore these amino acids could not have been
the cause for such a great drop in the CD4 count
between visits 2 and 3. Although the sequence did have
a point mutation from F20 to L20. Both amino acids are
hydrophobic and would fit into the hydrophobic pocket
on the ELC2 loop. Since the L20 is smaller than the F20
it is possible to postulate that the binding affinity could
be greater due to less steric hindrance. The second and
most profound mutation that occurred was the change
from S11 to R11. Research has shown that the serine is
preferred in the CCR5 binding where as the arginine is
preferred do to polar binding with other negatively
charged amino acids in the ECL2 of the CXCR4
receptor. The change in affinity from the CCR5 to the
CXCR4 has shown to be more lethal to HIV patients as
apposed to binding affinity to CCR5 solely. This is a
possible explanation as to why patient 10 was a rapidprogressor.
CCR5 and CXCR4
Receptor
Figure 2: Sequence and location of the amino acids for CCR5
The CCR5 and CXCR4 7 transmembrane receptors
that are utilized by the V3 loop of the gp120 to bind
the HIV virus to the T-cell. The extra cellular Nterminal domain is interacts with the stem regions of
the gp120 V3 loop and nonspecific binding from the
β19 strand of the gp120. The binding of the β19
strand is specific for CCR5 recognition. Research
has shown that S11 is specific for CCR5 biding
where as R11 is specific for CXCR4 binding. There
are many additional intermolecular interactions with
the V3 loop (R9, R18, F20, GPG motif, and Y21) that
cause recognition and binding to the receptors from
the gp120 V3 motif. The CCR5 has an overall
hydrophilic N-terminal and ECL2. Although within
the ECL2 there is an area of 5 amino acids, KEGLH,
which are hydrophobic and polar located towards
the membrane where the β hairpin turn may interact.
There are different interactions employed by the
CXCR4 receptor. The CXCR4 receptor has 3
aspartic acids that interact with the β hairpin loop
creating a hydrophobic pocket for the F20 to
interact. The two receptors are very similar in
function and structure although there are subtle
differences that allow the V3 loop to be specific to
either the CCR5 or CXCR4 receptor.
Non-progressor
Patient 12
Figure 3: The amino acid sequence of the overall mutational changes that occurred in patient 12 and patient 10. The 3 domains and secondary
structures are coordinated on the figure with the colored bars.
Figure 4: A point mutation study performed on the V3 loop measuring the binding affinities to
the CCR5 and CXCR4 receptors.
References
Markham, Richard (1998) Proc. Natl. Acad. Sci. 95, 12568-12573.
Catasti, Paolo (1995) J. Biol. Chem. 270, 2224-2232.
Cormier, Emmanuel (2002) J. Virol. 76, 8953-8957.
Siciliano, Salvatore (1999) J. Biol. Chem. 274, 1905-1913.
Basmaciogullari , Stephane (2002) J. Viol. 76, 10791-10800.
Suphaphiphat, Pirada (2003) J. Virol. 77, 3832-3837.
Cilliers, Tonie (2003) J. Virol. 77, 4449-4456.
Patient 12 was considered to be a non-progressor in
the study done in Baltimore MD. This patient was
considered a non-progressor by not observing a CD4
count drop throughout the duration of the study. After
transcribing all of the mutant gp120 V3 loops that
were obtained from the study, there were no point
mutations that occurred within the overall sequences.
For the purposes of this poster the V3 loop was
examined. The amino acid sequence closely
resembled that of the R5 sequence. Looking closely
at the sequence, the selected amino acids appear to
be specific for CCR5 binding and not CXCR4 binding.
Looking at the point mutation study done in figure 4
the amino acids that appear to significantly affect the
binding of V3 loop to CCR5 are still conserved (R11
and I26). Although a point mutation that is present
throughout the entire study for this patient is a change
from H13 to a P13. This amino acid is located in the
middle of a β strand. The phi and psi angles needed
to form a correct β strand are not able to be obtained
with a P13 mutation. Therefore the 3 dimensional
structure of the V3 loop is not oriented correctly with
the ECL2 and crown region of the V3 loop, thus not
allowing the binding to both the CCR5 and CXCR4.
The CD4 count would not be affected due to the lack
of the V3 loop to bind to the 2 receptors.
Tom Conrad Maugans Seegar
Ursinus College
Collegeville PA 19426