1. Loyola Marymount University
More Frequent Nonconservative Amino Acid Substitutions in the gp120 V3 Region in Patients with Rapid CD4+ T Cell Decline Versus Patients with High CD4+ T Cell Variability
Kevin Paiz-Ramirez
Janelle N. Ruiz
Biology
Department of Biology
Loyola Marymount University
March 20, 2010

2. Outline
Entry of HIV-1 involves binding of gp120 to CD4 receptor and co-receptors
Are there structural differences in V3 region of HIV-1 gp120 between participants with rapid CD4+ T cell decline versus patients with high CD4+ T Cell variability?
Results: comparing our groups
Diversity and divergence of gp120 V3 region amino acid sequences is higher in linear progresors versus high variability subjects
There are differences in secondary structure predictions between the two groups
Amino acid changes take place on the surface of gp120 and near the co-receptor binding site
Post-translational modifications linear progressors occur near or on the co-receptor binding site

3. The Entry of HIV Involves Specific Interactions between gp120, CD4, and the Co-receptor
Kwong et al. (1998) solved the crystal structure of
gp120 (red)
CD4 (yellow)
An antibody that binds similarly to the co-receptor (blue and aqua)
Gp120 is crucial for fusion of HIV-1 to cell surface
Positioning
Timing
CD4 binding induces conformational change in gp120
The envelope glycoprotein gp120 (red)
CD4 glycoprotein (yellow)
An antibody that binds similarly to the co-receptor (blue and aqua)

4. Structure of gp120 Core Kwong, et, al 1998
Main message of slide and put that as the title, showing specific secondary structures and how they highlight the location of the v3 loop.
Kwong, et, al 1998

5. Previous investigation revealed differences in diversity and divergence in DNA sequence of gp120 V3 variants
Patients with high CD4 decline had greater diversity and divergence in DNA sequence of gp120 V3 variants than patients with high variability in CD4 count
Are there amino acid changes that lead to structural differences in gp120 between patients with a rapid CD4+ T cell decline versus patients with high CD4+ T Cell variability?
Does this translate to differences in protein sequence and structure?
Linear Progressors
High Variability

6. Differences in Amino Acid Sequences were observed between the Linear and High Variability Groups
Multiple sequence alignments based on amino acid sequences were generated for each subject using the clones from the first four visits
Determined consensus sequence for each subject
Determined the sequence(s) with most differences from consensus sequence for each subject
Predicted secondary structures of sequences using PSIPRED
4. Performed Multiple Sequence alignments for sequences
5. Investigated sites of possible post-translational modifications using PROSITE

7. High Variability group has fewer differences between sequences and less divergence in sequences
Subject 12
Multiple Sequence Alignments
See less amino acid sequences in total and less major amino acid changes between sequences (purple) in High Variability group that Linear progressors
Tables: Represent Major Amino Acid Differences between Consensus Sequence and Sequence with most differences b/w Consensus Sequence 7

8. High Variability group has fewer differences between sequences and less divergence in sequences
Subject 8 D

9. High Variability group has fewer differences between sequences and less divergence in sequences
Subject 6

10. Linear Progressors had More Nonconservative Substitutions
Subject 4
Many more sequences and many more purple major amino acid changes, shown in chart 10

11. Linear Progressors had More Nonconservative Substitutions
Subject 10

12. Structure-Based Sequence Alignment of Core Gp120
Gp120 sequence from Kwong et al.

13. Predicted Secondary Structures of the gp120 V3 Region shows six beta sheets and one alpha helix

14. High Variability shows six beta sheets and one alpha helix
Subject 6
Subject 8
Subject 12

15. Linear Progressors show six beta sheets and one or two alpha helices
Subject 4
Subject 10

16. High Variability Participants had Amino Acid Substitutions Occur on the Protein Surface
Binding Site
*Black 1: Coil region, nonpolar, hydrophobic  Polar Basic
*Black 2: Coil Region, Polar BasicPolar, unchargednonpolar, hydrophobic
*Black 3: Helix 2, Nonpolar, hydorphobicPolar Basic
*Black 4: Coil Region, Polar, unchargednonpolar, hydorophobic
Key:
Blue: Single, fully conserved residues
Green: Conservation of strong groups
Purple: Conservation of weak groups
Black: No consensus

17. Linear Progressor Participants Had a Greater Number of Nonconservative Amino Acid Substitutions Located on Binding Sites
Binding Site
Surface
*Black 1: Beta Sheet 11, polar uncharged nonpolar, hydrophobic
*Black 2: Coil Region, Polar basic nonpolar, hydrophobic
*Black 3: Helix 2, polar uncharged nonpolar, hydrophobic
*Black 4: Coil Region, Polar BasicPolar, uncharged
1st black coil  N-binding site
2nd black coil on Betasheet
3rd black coil On the surface not on a binding site
4th black coil binding site
5th black coil on the surface not on a binding sit
Key:
Blue: Single, fully conserved residues
Green: Conservation of strong groups
Purple: Conservation of weak groups
Black: No consensus

18. Linear Progressor Participants Have Greater Major Amino Acid Changes on Protein Binding Sites
*Black 1: Coil Region, nonpolar, hydrophobic & Polar Basic  Polar Acidic
*Black 2: Beta Sheet 11, Nonpolar Basic  Polar Uncharged
*Black 3: Coil Region, Polar basic nonpolar, hydrophobic
*Black 4: Coil Region, Polar, uncharged  Polar Basic
*Black 5: Coil Region, random
*Black 6: Helix 2, Polar, uncharged & Polar Basic  nonpolar, hydrophobic
*Black 7: Helix 2, nonpolar, hydrophobic & Polar Basic  Polar, uncharged & Polar Acidic
*Black 9: Coil Region, Polar Basic  Polar Uncharged
*Black 10: Coil Region, random
Key:
Blue: Single, fully conserved residues
Green: Conservation of strong groups
Purple: Conservation of weak groups
Black: No consensus

19. Linear Progessor Participant Displaying Nonconservative
Amino Acid Substitution Located on Binding Site

20. Sites of Post Translational Modification: Linear Progressors Have Slightly Greater Post Translational Modifications than High Variability

21. The Linear Progressor group was different from the High Variability group on a number of different measures
Linear progressors
had greater number of amino acid sequence differences
greater amount of nonconserved amino acid substitutions
had greater number of major amino acid changes in CD4-binding regions
showed greater post-translational modifications
This could indicate greater structural differences in linear progressors
Further studies could examine protein structural differences variants within gp41 region
Linear progressors had greater number of amino acid sequences (diversity)
Amino acid changes lead to larer sturctural differences in the protein

22. Pancera et al. (2010) examined the region where gp120 interacts with gp41
This region had not been explored by earlier studies (Kwong et al)
gp120 elements proximal to gp41 completed a 7 stranded Beta sandwich
Facilitated movement between outer domain and gp41
Kwong et al. also observed a conformational change with the virus in the v3 region
This corroborates with Pancera’s observed changes in the gp41

23. Conclusion
Entry of HIV-1 involves the binding of gp120 to CD4 receptor and co-receptors
There are structural differences in the V3 region of HIV-1 gp120 between participants with rapid CD4+ T cell decline and patients with high CD4+ T Cell variability
The diversity and divergence of gp120 V3 region Amino Acid sequences is higer in linear progressors
Presented differences in secondary structure between linear and high variability
Amino acid changes take place on the surface of gp120 and near the co-receptor binding site
Post-translational modifications linear progressors occur near or on the co-receptor binding site
Pancera et al & Kwong et al observed conformational changes in the gp41 and V3 region 23

24. References
Kwong PD, Wyatt R, Robinson J, Sweet RW, Sodroski J, and Hendrickson WA. Structure of glycoprotein in complex with the CD4 receptor and a neutralizing human antibody. Nature 1998 Jun 18; 398(6686)
Pancera M, Majeed S, Ban YE, Chen L, Huang CC, Kong L, Kown YD, Stuckey J, Zhou T, Robinson JE, Schief WR, Sodroski J, Wyatt R, and Kwong PD. Structure of HIV-1 gp120 with gp41-interactive region reveals layered envelope architecture and basis of conformational mobility. Proc Natl Acad Sci USA 2010 doi: