Transcript Specific changes in the primary structure of rapid and non

Specific changes in structure
of HIV-1 rapid progressors and
nonprogressors
BioInformatics Lab
Tuesday, March 23, 2010
Kristoffer Chin
Salomon Garcia
Michael Piña
Outline
• Background
– Introduction on HIV structure
– Our question about HIV structure
• Methods
– Analyzing the amino acid structure
• Results & Discussion
• Comparing our finding to a more recent
article
• References
HIV attacks the immune system
• The function of an HIV-1 virus is to attach
to the CD4 T-cell with the gp120
http://www.bijvoet-center.nl/cpc/research/HIVEnv/Env.jpg
Altered function in rapid and
nonprogressors
• Rapid progressors increase in viral
load which causes rapid decline of
CD4 T-cells
• Nonprogressors take a longer time in
the decline of CD4 T-Cells and
sometimes increases
• More basic charged amino acids
should be found in rapid progressors
(Hill & Hernandez, 2006)
Is there a difference in the primary structure of
rapid and nonprogressors which ultimately
affect secondary and tertiary forms altering
the function of the virus?
• H₀ – There will not be any difference
in the primary structure of the HIV
gp120 env region in Markham’s
experimental subjects
• H₁ – There is a difference found in
the primary structure of the HIV
gp120 env region in Markham’s
experimental subjects
Choosing the subjects, visits,
and clones
• Rapid progressors
– Subjects 4 & 10
• Nonprogressors
– Subjects 12 & 13
• We took 4 visits per subject
• We then took 4 clones per visit for a total of 64
clones
• Clones were chosen based on max distances
Methods for generating
sequences and figures
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Amino Acid Sequences were obtained
– UniProt for the gp120 protein
– Bedrock for the sequences corresponding to the Markham et al. article
Biology Workbench
– CLUSTALW for sequence alignment
– CLUSTALDIST to find min and max distance
– BOXSHADE for sequences alignment comparisons
ProtParam
– Primary structure analysis
Predicting Secondary Structures
Cn3D to compare crystal structures
– Structure files obtained from NCBI Structure Database
Multiple sequence alignments illustrate
stronger conservation in nonprogressors
ProtParam tool used to analyze primary
structures of rapid progressors
Subject 4
Subject 10
No significant differences found in the amino
acid composition in rapid and nonprogressors
Subject 12
Subject 13
Predicting the secondary structure
of the protein sequences
• http://bioinf4.cs.ucl.ac.uk:3000/psipred
• Secondary structures were predicted for
the entire gp120 and gp41 region with the
data obtained from UniProt
• Secondary structures were predicted for
the chosen subjects
– Consensus strand from subjects 4 & 10
– Consensus strand from subjects 12 & 13
Predicted secondary structure for
gp120 + gp41
Predicted secondary structure of the V3
region based on consensus strands
are very similar
Subjects 4 & 10
Subjects 12 & 13
Crystal structure of V3 loop (1GC1) is in complex
with CD4 and neutralizing antibody
• Minor changes in
the amino acid
sequence are not
changing the
secondary
structure of the
protein
V3 Region
Differences in the sequences between
rapid progressors and nonprogressors
• We did not see any notable amino acid
changes that would alter the structure or
function of the V3 region
• We are accepting our null hypothesis
– There will not be any difference in the primary
structure of the HIV gp120 env region in
Markham’s experimental subjects
Structural basis of immune evasion at the
site of CD4 attachment on HIV-1 gp120
• Lei Chen et al. (2009)
• Although gp120 readily binds to antibodies, most
are unable to neutralize HIV-1
• Crystal structures in complex with two poorly
neutralizing antibodies show that the
conformations are not compatible with the viral
spike
• Thusly, it was concluded that HIV-1 is resistant
to all but the most accurately targeted antibodies
Conclusion
• Perhaps change in the amino acid
structure of the HIV-1 virus is not as
important as finding antibodies that
successfully neutralize the virus
• Future studies can be done to determine
properties of successful antibodies
References
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Stanfield et al. Recurring conformation of the human immunodeficiency
virus type 1 gp120 V3 loop. Virology (2003) Oct 10; 315(1) 159-73
Markham RB, Wang WC, Weisstein AE, Wang Z, Munoz A, Templeton A,
Margolick J, Vlahov D, Quinn T, Farzadegan H, and Yu XF. Patterns of HIV1 evolution in individuals with differing rates of CD4 T cell decline. Proc Natl
Acad Sci U S A 1998 Oct 13; 95(21) 12568-73
Hill MD and Hernandez W. Nucleotide and amino acid mutations in human
immunodeficiency virus corresponding to CD4+ decline. Arch Virol 2006
Jun; 151(6) 1149-58
Kwong PD, Wyatt R, Robinson J, Sweet RW, Sodroski J, and Hendrickson
WA. Structure of an HIV gp120 envelope glycoprotein in complex with the
CD4 receptor and a neutralizing human antibody. Nature 1998 Jun 18;
393(6686) 648-59.
http://www.bijvoet-center.nl/cpc/research/HIVEnv/Env.jpg