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 398.01 Department of Biology Loyola Marymount University March 20, 2010

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

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)

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

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

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

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

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

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

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

Linear Progressors had More Nonconservative Substitutions Subject 10

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

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

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

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

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

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

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

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

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

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

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

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

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) 648-59. 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:10.1073