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A Mutational Investigation of an HIV Patient’s GP120 Glycoprotein and it’s Implications on
CD4 Binding
Salita Kaistha
Usrinus College, Collegeville PA 19426
November 28, 2003
DNA Results Continued
Background
HIV is a deadly virus killing more than 3 million people a year. This fact alone motivates researchers
to learn as much as possible about the virus, its structure, and it’s entry into cells, in hopes of eventually
developing a cure. Viewed under a microscope an HIV particle would look like this:
Structural Results
One advantage that HIV has over our immune system is its rapid rate of mutation. There are both
synonymous and non-synonymous mutations. Synonymous mutations are those which involve changes
in the nucleotide sequence, but still produce the same amino acid sequence. Non-synonymous
mutations are those which change the nucleotide sequence and the amino acid sequence.
Clone 3 from Visit 1 is what I consider to be the original strain. All other clones are variations of this
strain. Clone 4 from Visit 4 is an example of a synonymous mutation, for there is a change in the DNA
sequence (1 nucleotide), but the resulting amino acid sequence is the same (Fig. 1). Visit 1 clone 1 is
an example of a non-synonymous mutation. There was a change in one nucleotide of the DNA
sequence, however this time the DNA sequence produces a different amino acid sequence (Fig. 1).
Amino Acid Results
An HIV particle is approximately 0.0001 mm. An HIV particle consists of two main parts, the inner
core and the viral membrane. This viral membrane contains two main glycoprotein's, GP120 and GP41.
GP120 allows the HIV particle to bind to CDR+ T cells of our immune system. GP41 facilitates membrane
fusion.
Due to GP120’s involvement in cell recognition and binding, it is of great interest to researchers.
GP120 is encoded for by 500 amino acids, but only a small portion of these are involved in binding to CD4.
In fact, GP120 interactions come from 6 fragments located on the V1/V2 stem, Loop D, B15-alpha15
excursion, B20-B21 hairpin, B23, and the B24-alpha5 connection. This coincides with the general principle
that much of a protein’s secondary structure is simply needed to provide a scaffold, and it is the loops that
allow the protein to function. The progression of HIV is monitored by an individual’s CD4 T cell count.
Prior Research
This study was conducted using research and data collected by Markham et al. Markham et al.
followed 15 patients for anywhere from 1 ½ - 4 ½ years. They monitored the number of clones of HIV
within the individual and the individuals CD4 T cell count. The number of clones per visit ranged
anywhere between 2 and 18. The researchers gathered over 666 DNA sequences.
Methods and Materials
This study was conducted using the data gathered by Markham et al. The study focused on patient #
11, a rapid progressor for HIV. Data was collected for the last two years of the patient’s life. Resources
used include those found at PDB, NCBI and ExPASy.
DNA Results
Visit
Clone
Represents
3
7 22 27 29 33 37 42 51 52 56 66 70 71 78 79 86 91
VISIT #2
Clone #1
Clone #2
Clone #3
Clone #4
Clone #5
Clone #6
7 22 27 29 33 37 42 51 52 56 66 70 71 78 79 86 91
VISIT #3
Clone #1
Clone #2
Clone #3
Clone #4
Clone #5
Clone #6
Clone #7
Clone #8
Clone #9
Clone #10
7 22 27 29 33 37 42 51 52 56 66 70 71 78 79 86 91
VISIT #4
Clone #1
Clone #2
Clone #3
Clone #4
Clone #5
Clone #6
Clone #7
Clone #8
Clone #9
Clone #10
7 22 27 29 33 37 42 51 52 56 66 70 71 78 79 86 91
K
E
E
K
E
E
K
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
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E
E
E
E
E
G
E
K
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
N
T
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
A
T
T
T
T
T
T
T
T
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
Y
N
N
N
Y
N
N
Y
N
Q
Q
Q
Q
Q
Q
Q
Q
Q
Q
Q
Q
Q
I
I
I
I
I
I
I
I
I
I
I
I
I
Q
P
Q
Q
Q
Q
Q
Q
Q
Q
I
I
I
I
I
V
I
I
I
I
Q
Q
Q
Q
Q
Q
Q
Q
Q
Q
I
I
I
I
I
I
I
I
I
I
T
T
T
T
T
T
T
T
T
T
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T
T
T
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T
T
T
T
T
T
A
T
T
T
T
T
T
T
T
T
T
T
G
G
G
G
G
G
G
G
G
G
G
R
G
G
G
G
G
E
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
N
N
N
N
N
N
N
N
N
N
N
N
N
N
D
N
N
N
N
N
N
N
N
D
N
D
N
N
N
N
N
N
N
R
R
R
E
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
G
R
R
R
R
R
G
R
R
N K R K L V
N K R K L V
N K R K L V
N K R K L V
N K G K L V
N K R R L V
D K R K L V
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
K
K
R
K
K
K
K
K
K
K
K
K
K
K
K
K
K
K
K
K
K
K
K
K
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K
R
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R
R
R
R
R
R
R
R
K
K
K
R
K
K
K
K
K
K
K
K
K
K
K
K
L
L
L
L
L
L
P
L
L
L
P
L
L
L
L
L
orginal strain
22
29
33
37
V
V
V
V
V
V
V
V
V
V
51
56
66
70
71
78
79
86
22
27 29
33
37
42
51
56
66
70 71
78 79
86
1
1
EV IIRS K NF S NNA K NIIV Q L N ES V V INCT RPDNT IK Q RIIHIG PG RPF Y T T G IK G NIRQ A HCNV S RG Q W NK T L EQ V V RK L REQ Y G L NK T IV F K Q PI
1
2
EV IIRS ENF S NNA K NIIV Q L N ES V V IT CT RPDNT IK Q RIIHIG PG RPF Y T T G IK G NIRQ A HCNV S RG Q W NK T L EQ V V RK L REQ Y G L NK T IV F K Q PI
1
3
EV IIRS ENF S NNA K NIIV Q L N ES V V INCT RPDNT IK Q RIIHIG PG RPF Y T T G IK G NIRQ A HCNV S RG Q W NK T L EQ V V RK L REQ Y G L NK T IV F K Q PI
1
4
EV IIRS K NF S NNA K NIIV Q L N ES V V INCT RPDNT IK Q RIIHIG PG RPF Y T T G IK G NIRQ A HCNV S EG Q W NK T L EQ V V RK L REQ Y G L NK T IV F K Q PI
1
5
EV IIRS ENF S NNA K NIIV Q L N ES V V INCT RPDNT IK Q RIIHIG PG RPF Y T T G IK G NIRQ A HCNV S RG Q W NK T L EQ V V G K L REQ Y G L NK T IV F K Q PI
1
6
EV IIRS ENF S NNA K NIIV Q L N ES V V INCT RPDNT IK Q RIIHIG PG RPF Y T T G IK G NIRQ A HCNV S RG Q W NK T L EQ V V RRL REQ Y G L NK T IV F K Q PI
1
7
EV IIRS K NF S NNA K NIIV Q L N ES V V INCT RPDNT IK Q RIIHIG PG RPF Y T T G IK G NIRQ A HCNV S RG Q W DK T L EQ V V RK L REQ Y G L NK T IV F K Q PI
7
22
27 29
33
37
42
51
56
66
70 71
78 79
86
4
1
EV IIRS ENF S NNA K NIIV Q L N ES V V INCT RPDNT IK Q RIIHIG PG RPF Y T T G IK G DIRQ A HCNV S RG Q W NK T L EQ V V RK L REQ Y G L NK T IV F K Q PI
4
2
EV IIRS ENF S NNA K NIIV Q L N ES V V INCA RPDY T IK Q RIIHIG PG RPF Y T T G IK G NIRQ A HCNV S G G Q W NK T L EQ V V RK L REQ Y G L NK T IV F K Q PI
4
3
EV IIRS ENF S NNA K NIIV Q L N ES V V INCT RPDNT IK Q RIIHIG PG RPF Y T T G IK G DIRQ A HCNV S RG Q W NK T L EQ V V K K L REQ Y G L NK T IV F K Q PI
4
4
EV IIRS ENF S NNA K NIIV Q L N ES V V INCT RPDNT IK Q RIIHIG PG RPF Y T T G IK G NIRQ A HCNV S RG Q W NK T L EQ V V RK L REQ Y G L NK T IV F K Q PI
4
5
EV IIRS ENF S NNA K NIIV Q L N ES V V INCT RPDNT IK Q RIIHIG PG RPF Y T T G IK G NIRQ A HCNV S RG Q W NK T L EQ V V RK L REQ Y G PNK T IV F K Q PI
4
6
EV IIRS ENF S NNA K NIIV Q L N ES V V INCT RPDY T IK Q RIIHIG PG RPF Y T T G IK G NIRQ A HCNV S RG Q W NK T L EQ V V IK L R EQ Y G PNK T IV F K Q PI
4
7
EV IIRS ENF S NNA K NIIV Q L N ES V V INCT RPDNT IK Q RIIHIG PG RPF Y T T G IK G NIRQ A HCNV S RG Q W NK T L EQ V V RK L REQ Y G L NK T IA F K Q PI
4
8
EV IIRS ENF S NNA K NIIV Q L N ES V V INCT RPDNT IK Q RIIHIG PG RPF Y T T G IK G NIRQ A HCNV S G G Q W NK T L EQ V V K NL REQ Y G L NK T IV F K Q PI
4
9
EV IIRS ENF S NNA K NIIV Q L N ES V V INCT RPDY T IK Q RIIHIG PG RPF Y T T G IK G NIRQ A HCNV S RG Q W NK T L EQ V V K K L REQ Y G L NK T IV F K Q PI
Figure 2. The amino acids found at selected positions for all of the clones found at all visits for patient 11. The selected positions are those which experienced
a mutation at any time during the two years. The mutated amino acids are highlighted according to the following color scheme: yellow for Glycine’s, green for
hydrophobic, pink for negatively charged, blue for positively charged and purple for polar amino acids.
Figure 2 portrays only the amino acid positions that encountered changes over the 2 years. There are
3 predominant changes taking place. These changes include converting the charge (+  -), converting to
a smaller amino acid (a.a. G), or a conservative change (+  +).
Visit #
# of
Clones
# a.a. positions
altered (this
visit)
Total # a.a.
positions altered
(since 1st visit)
# synonomous
mutations
# nonsynonomous
mutations
# of strains with
1 a.a. position
changed
# of strains with
2 a.a. positions
changed
# of strains with 3
a.a. positions
changed
CD4+ Tcell count
1
7
6
6
0
7
4
2
0
753
2
6
3
9
3
3
3
0
0
600
3
10
7
14
4
6
4
1
2
270
4
9
8
17
2
8
3
2
3
175
References
Kwong, P., Wyatt, R., and J. Robinson. (1998) Structure of an HIV gp120 envelope glycoprotein in complex
with the CD4 receptor and a neutralizing human antibody. Nature 393, 648-659.
Markham, R., Wnag, W. and A. Weisstein. (1998) Patterns of HIV-1 evolution in individuals with differing
rates of CD4 T cell decline. Proc. Natl. Acad. Sci. 95, 12568-12573.
The Protein Data Bank. (2003)
http://www.rcsb.org/pdb/cgi/explore.cgi?job=graphics&pdbId=1G9M&page=&pid=151221070341180.
The Molecules of HIV. (2003) http://www.mcld.co.uk/hiv/?q=HIV%20virus%20particle.
Aids in the World. (2003) http://www.yale.edu/yaw/world.html.
TTAGAACAGGTAGTTAGAAAATTAAGAGAACAATATGGACTGAATAAAACAATAGTCTTTAAGCAACCCATA
4
4
synonomous
mutation
As the disease progresses there is a simultaneous drop in CD4+ T cell counts and an increase in the
number of mutations.
22
GAGGTAATAATTAGATCTGAGAATTTCTCAAACAATGCTAAAAACATAATAGTACAGCTGAATGAATCTGTA
27
29
33
37
42
GTAATTAATTGTACAAGACCCGACAACACTATAAAACAAAGGATAATACATATAGGACCAGGGAGACCATTC
51
56
66
70
71
Over two years there are 32 strains and only 17 amino acid positions altered over the sequence of 96
amino acids (less than 18%).
TATACAACAGGAATAAAAGGAAATATAAGACAAGCACATTGTAACGTTAGTAGAGGACAATGGAATAAAACT
78
79
86
91
TTAGAACAGGTAGTTAGAAAATTAAGAGAACAATATGGACTAAATAAAACAATAGTCTTTAAGCAACCCATA
7
1
1
non-synonomous
mutation
22
The percentage of non-synonymous mutations increases as the disease progresses.
GAGGTAATAATTAGATCTAAGAATTTCTCAAACAATGCTAAAAACATAATAGTACAGCTGAATGAATCTGTA
27
29
33
37
42
GTAATTAATTGTACAAGACCCGACAACACTATAAAACAAAGGATAATACATATAGGACCAGGGAGACCATTC
51
56
66
70
There are never more than 3 amino acid positions altered per strain of HIV, regardless of the visit number.
The number of strains with an increasing number of mutations per strain increases as the disease
progresses.
71
TATACAACAGGAATAAAAGGAAATATAAGACAAGCACATTGTAACGTTAGTAGAGGACAATGGAATAAAACT
78
79
86
91
TTAGAACAGGTAGTTAGAAAATTAAGAGAACAATATGGACTGAATAAAACAATAGTCTTTAAGCAACCCATA
Visit
Clone
Represents
Resulting Amino Acid Sequence
7
1
4
1
3
4
1
original strain
synonomous
non-synonomous
22
27 29
33
37
42
51
56
66
70 71
78 79
86
91
E V IIR S E N F S N N A K N IIV Q L N E S V V IN C T R P D N T IK Q R IIH IG P G R P F Y T T G IK G N IR Q A H C N V S R G Q WN K T L E Q V V R K L R E Q Y G L N K T IV F K Q P I
E V IIR S E N F S N N A K N IIV Q L N E S V V IN C T R P D N T IK Q R IIH IG P G R P F Y T T G IK G N IR Q A H C N V S R G Q WN K T L E Q V V R K L R E Q Y G L N K T IV F K Q P I
E V IIR S K N F S N N A K N IIV Q L N E S V V IN C T R P D N T IK Q R IIH IG P G R P F Y T T G IK G N IR Q A H C N V S R G Q WN K T L E Q V V R K L R E Q Y G L N K T IV F K Q P I
Figure 1. Codons encoding amino acid positions that were mutated over the 2 years are highlighted in blue. The codons containing the point
mutations are highlighted in pink.
91
As mentioned earlier, GP120 interacts with CD4 through 6 different fragments or portions on
GP120. The DNA sequences obtained for patient 11 encode for one of these 6 fragments, that is the
V1/V2 stem (Fig. 5). I have located the 6 amino acids that are critical for this interaction. They are
AHCNVS. Due to the fact the HIV must bind to CD4 in order to infect an individual’s cells, these 6
amino acids are critical to the virus’s infection and survival. Thus, logically, these 6 amino acids
would be conserved, and this is precisely what was discovered. This crucial conserved sequence,
AHCNVS, is highlighted in green in Figure 4.
R K L V
R K L V
K K L V
R K L V
R K P V
I K P V
R K L A
K N L V
K K L V
R K L V
91
7
91
These sequences demonstrate the fact that these mutations are not occurring throughout the
entire sequence, but rather at specific portions of the amino acid sequence over the 2 ½ years (Fig.
4). In other words certain portions are subject to mutations, where as other portions are conserved
and experience no mutation over the two year period.
Figure 3. This chart highlights the information found in the amino acid sequences listed above and also gives the CD4+ T cell counts for the patient at each
visit.
TATACAACAGGAATAAAAGGAAATATAAGACAAGCACATTGTAACGTTAGTAGAGGACAATGGAATAAAACT
7
Figure 5. This is a schematic representation of a portion of GP120 that interacts with CD4. It shows the V1/V2 stem on HIV’s GP120, which is encoded for by
patient 11’s DNA sequence. The green box highlights 6 amino acids on the V1/V2 stem that are necessary for binding to CD4. The pink box highlights the
fragment of CD4 that the V1/V2 stem interacts with.
42
GTAATTAATTGTACAAGACCCGACAACACTATAAAACAAAGGATAATACATATAGGACCAGGGAGACCATTC
Amino Acid Sequence
V
V
V
V
V
V
GAGGTAATAATTAGATCTGAGAATTTCTCAAACAATGCTAAAAACATAATAGTACAGCTGAATGAATCTGTA
27
Clone
Figure 4. This chart presents the 96 amino acid sequences for the envelope gene of the GP120 glycoprotein of HIV in the various clones of patient 11 at visits 1
and 4. Amino acids at positions were subject to mutation at any time during the two years are highlighted in blue. Amino acids there were mutated are
highlighted in pink. The amino acids highlighted in green represent a region on the V1/V2 steam that is critical for GP120 to bind to CD4.
DNA Sequence
7
1
VISIT #1
Clone #1
Clone #2
Clone #3
Clone #4
Clone #5
Clone #6
Clone #7
Visit
Acknowledgements
I would like to thank Dr. Roberts, Tom Seegar, Derese Getnet and Drew Foy for all their
help in completing this poster.