Residue Sequence and Structure in the Re evaluation the Categorization of HIV Progression in Subjects Based

on CD4 T cell Decline Rates

Angela Garibaldi & Ryan WillhiteLoyola Marymount University

BIOL 398-01March 23, 2010

Outline

• Review of previous experiment using CD4 decline rates

• Structure based hypothesis• Methods and programs used• Results• Comparison to More Recent Studies• References

Recap of past experiment

• Evaluated the categorization of progressors based on CD4 decline rates

• Analyzed two moderate progressors (6,7) with rates comparable to non-progressor 13 and rapid progressor 10.

• Based on divergence and diversity subject 6 did not act as non-progressor

• Subject 7 acted like a rapid progressor.

Hypothesis

• Subject 7’s protein structure and properties will be more similar to Rapid Progressor Subject 10 than to Moderate Progressor 5.

• Looking at protein sequences and structure is a more efficient method of testing our CD4 T-cell decline rate categorization than nucleotide sequence.

Methods

• Create phylogenetic trees based on amino acid sequence using Biology Workbench

• Use ProtPram to analyze residue composition in subjects 6,7,5,10,13

• Select 7, 10, and 5 for time point analysis– All clones used for selected visits

• Use PSIPRED to analyze secondary structures of 7, 10 , 5, 13

Phylogenetics based on amino acid sequence

10 vs 5 10 vs 7 5 vs 7

Subject 6 v 13

Subjects 6 v 7

Subjects 13 v 5

Subjects 6 v 5

ProtParam

Physico-chemical properties of a protein using ExPASy server

Subject 5 Properties

Subject 6

Subject 7

Subject 10

Overall residue composition

•No significant pattern in Negative/Positive Charged residues•Rapid Progressor Subject 10 is the only subject with Asn as its most prevalent residue

Residue composition over time

•Asn (Asparagine) is a carboxamide and is not an essential amino acid

•Often found at the beginning and ends of helices•Provides key sites for N-linked glycosylation

Residue composition over time

• All clones from selected visits were used.• Subject 10, Rapid Progressor shows Asn as its

prevalent residue over time.• Subject 7, Moderate Progressor begins with Asn as

prevalent residue• Subject 10 showed sudden jump in Arg %. This may

be an artifact.• Negative/Positive Charged residue ratio conserved

over time

Secondary StructureSubject 7 Visit 5

Subject 10 Visit 6

Subject 5 Visit 5 Subject 13 Visit 6

No Difference!

Conclusion• The highest percent of amino acids found in the

moderates 5,6, and 7 is (Ile)

• The highest percent of amino acids found in the rapid 10 to compare is (Asn)

• These differences did not equate to significant secondary structure differences

• While looking at these compositions along with protein structure is a questionable method of progressor categorization, it provides insight regarding qualities of progressors.

More recent study by YS Ho et al

• N-linked Glycosylation (NGL) important in minimizing virus neutralizing antibody response

• Looked at env gp120 C2-V5 region (includes V3) in plasma, leukocyte and other compartments

• Significant variation in numbers of NGL sites between patients

• Found single cell/compartment-specific amino acid changes and differences in NLG patterns between plasma and blood leukocytes

Prediction of NGL sites on gp120

• Figure 2. YS Ho et al

V3 Region NGL sites

•V3 region has 1 clearly significant NGL site along with 2 lesser NGL sites

Conclusions in relation to YS Ho• The dominance of Asn % in Rapid Progressor amino

acid composition across all time points may lead to the increase of NGL sites

• Dominance of Asn % in Subject 7 during Time Point 1 suggests subject does share some Rapid Progressor qualities

• Increase in NGL sites or changes in NGL sites may be a mechanism of dealing with HIV-1 in its later stages– May influence viral recognition by host antibodies

Future Direction

•Analyze amino acid composition in larger number of Rapid Progressors for Asn dominance•Based on subject sequences, predict NGL sites per individual•Compare these with that of Non-Progressors•Analyze the folding patterns of the gp120 protein based on differences in the V3 region where Asn% is dominant vs Ile%.

References

• Ho YS, Abecasis AB, Theys K, Deforche K, Dwyer DE, Charleston M, Vandamme AM, and Saksena NK. HIV-1 gp120 N-linked glycosylation differs between plasma and leukocyte compartments. Virol J 2008 Jan 23; 5 14. doi:10.1186/1743-422X-5-14 pmid:18215327