Examination of Amino Acid Differences as a Means of Determining Functional Changes in HIV-1 Protein Sequences Chris Rhodes and Isaiah Castaneda Loyola Marymount University Department of Biology BIOL /2/11

Outline Amino acid sequence is related to protein function through structure and sidechain interactions Previous experiments indicate possible links between phylogenetic trees and Subjects’ immune response pattern Conserved residue changes between individual visits indicate potential functional differences in the protein sequences Observed functional differences between visits agree with previous conclusions. Subject 7 shows a Best Fit immune response and Subject 13 shows a Broad immune response According to Ogert et al. (2009) certain residue changes can result in immune cell resistance to infection by HIV-1 strains

Protein function is dependent on numerous structural effects of the amino acid sequence Protein structure and therefore function is affected by: Amino Acid Type – Acidic, Basic, Polar Uncharged, or Nonpolar Side Chain Interactions – Hydrophillic/Hydrophobic Interactions – Ionic Bonding – Cysteine Disulfide Bonding Significant changes in the amino acid sequence will have an effect on the function of the protein

Our previous study shows a link between patterns of phylogenetic trees and patterns of Subjects’ immune response Based on interpretations of the phylogenetic trees of the Subjects’ viral clones the pattern of each Subjects’ immune response was hypothesized Subject 13 showed a Broad immune response: Low Diversity and Low Divergence Little Branching Strong Selection Against Change Subject 7 showed a Best Fit immune response High Diversity and High Divergence One Prominent Branch Strong Selection Against Change from Best Fit Sequence Phylogenetic trees of amino acid sequences can be used to indicate possible functional differences

Experimental sequences were chosen based on phylogenetic trees of amino acid sequences Major divergence of amino acid sequences indicates possible functional differences between sequences Sequences were chosen in order to maximize possible functional differences and to be relevant to the previous study The chosen sequences for Subject 7 are: All visit 3 sequences Visit 4 sequences 1, 2, 3, 4, and 6 Visit 5 sequences 1, 3, 6, 7, 8, and 9

Experimental sequences were chosen based on phylogenetic trees of amino acid sequences Divergence of amino acid sequences indicates possible functional differences between sequences Subject 13 is hypothesized to show strong selection against change in function Due to the divergence and disappearance of the Visit 4 sequences it can be assumed that Visit 4 may constitute a functional change The Visit 5 sequences were chosen to act as a control representing the root sequences The chosen sequences for Subject 13 are: All of Visit 4 sequences All of Visit 5 sequences

Outline Amino acid sequence is related to protein function through structure and sidechain interactions Previous experiments indicate possible links between phylogenetic trees and Subjects’ immune response pattern Conserved residue changes between individual visits indicate potential functional differences in the protein sequences Observed functional differences between visits agree with previous conclusions. Subject 7 shows a Best Fit immune response and Subject 13 shows a Broad immune response According to Ogert et al. (2009) certain residue changes can result in immune cell resistance to infection by HIV-1 strains

A comparison of Subject 13’s Visit 4 and 5 amino acid sequences shows two major conserved changes Conserved changes are residue changes the are consistent throughout all the sequences of a visit Conserved changes are most likely to indicate functional differences between different visits Between Visits 4 and 5 there are two conserved residue changes: 1.G  R 2.R  G

R  G and G  R changes constitute major structural differences Arginine (R) Glycine (G) Arginine contains a large, hydrophilic, and positively charged side chain Glycine contains a very small, hydrophobic, and uncharged side chain The difference between the two amino acids would have a major effect on protein structure and therefore function

A comparison of the selected Subject 7 amino acid sequences shows multiple conserved changes Between the selected sequences there are a total of eleven conserved changes Of these eleven conserved changes nine of them are major changes and constitute potential functional change in the protein

Multiple conserved residue changes of Subject 7’s alignment show functional significance Visit 4 Conserved Changes: 1.L  P: Proline is a helix breaker and is inflexible compared to Leucine 2.A  T: Hydrophobic Nonpolar to Hydrophillic Polar Uncharged 3.K  N: Hydrophillic Positive Charge to Hydrophillic Polar Uncharged 4.S  P: Small Hydrophillic Polar Uncharged to Hydrophobic Nonpolar 5.N  D: Hydrophillic Polar Uncharged to Hydrophillic Negative Charge 6.K  Q: Hydrophillic Positive Charge to Hydrophillic Polar Uncharged 7.E  G: Polar Negatively Charged to Nonpolar Hydrophobic Visit 5 Conserved Changes 1.V  I: Hydrophobic Nonpolar to Hydrophobic Nonpolar – No functional change 2.S  P: Small Hydrophillic Polar Uncharged to Hydrophobic Nonpolar Visit 3 Conserved Changes 1.T  S: Hydrophillic Polar Uncharged to Hydrophillic Polar Uncharged – No functional change 2.A  T: Hydrophobic Nonpolar to Hydrophillic Polar Uncharged

Outline Amino acid sequence is related to protein function through structure and sidechain interactions Previous experiments indicate possible links between phylogenetic trees and Subjects’ immune response pattern Conserved residue changes between individual visits indicate potential functional differences in the protein sequences Observed functional differences between visits agree with previous conclusions. Subject 7 shows a Best Fit immune response and Subject 13 shows a Broad immune response According to Ogert et al. (2009) certain residue changes can result in immune cell resistance to infection by HIV-1 strains

Conserved changes in Subject 13’s amino acid sequences indicate functional differences between Visits consistent with a Broad immune response pattern Based on the R  G and G  R changes between the Visit 4 and 5 sequences it is likely that the sequences of Visit 4 have a different function than those of Visit 5 The disappearance of Visit 4 in the phylogenetic tree could thus be explained by the functional difference between Visits 4 and 5 This agrees with the previous findings that Subject 13 uses a Broad immune response pattern that is selective against functional change Since the Visit 4 sequences represent a change in function from the Visit 5 sequences they were eradicated by the immune system

Conserved changes in Subject 7’s amino acid sequences indicate functional differences between Visits consistent with a Best Fit immune response pattern The multiple conserved changes between the Visit 4 sequences and the other two Visits indicate Visit 4 has a drastically different function than both the Visit 3 and Visit 5 sequences This agrees with the previous findings that Subject 7 uses a Best Fit immune response where Visit 4 represents the best fit sequence Since the Visit 3 sequences had such a divergent function from the Visit 4 sequences they were wiped out by the immune system which is selective against drastic change from the best fit sequence Based on this is it likely we would also see a disappearance of the divergent Visit 5 clones in a hypothetical Visit 6 sample

Outline Amino acid sequence is related to protein function through structure and sidechain interactions Previous experiments indicate possible links between phylogenetic trees and Subjects’ immune response pattern Conserved residue changes between individual visits indicate potential functional differences in the protein sequences Observed functional differences between visits agree with previous conclusions. Subject 7 shows a Best Fit immune response and Subject 13 shows a Broad immune response According to Ogert et al. (2009) certain residue changes can result in immune cell resistance to infection by HIV-1 strains

Research by Ogert et al. (2009) indicates that specific amino acid changes in the V3 loop corresponds to viral infectivity potential The function of the V3 loop can be affected by six specific amino acid changes When all six changes are present in the V3 sequence the virus cannot infect CD4 cells and replicate These findings show the link between amino acid residue change and viral functionality It is possible that minute changes in the amino acid sequence can render a protein non-functional For our experiment we assume all proteins are functional but it is possible that this is not the case If not, the disappearance of certain visits may then be due to mutations causing non-functionality

Summary Previous research shows a link between phylogentic patterns and Subject immune response patterns Changes in the amino acid sequence of a protein can affect functionality When comparing amino acid sequences conserved changes can be considered to be functionally relevant Functional differences between observed sequences confirm previous hypothesis about Subjects’ immune response type Subject 7 shows a Best Fit immune response pattern Subject 13 shows a Broad immune response pattern

Acknowledgements Kam D. Dahlquist, Ph. D Ogert, Robert, Yan Hou, Lei Ba, Lisa Wojcik, Ping Qiu, Nicholas Murgolo, Jose Duca, Lisa Dunkle, Robert Ralston, and John Howe. "Clinical resistance to vicriviroc through adaptive V3 loop mutations in HIV-1 subtype D gp120 that alter interactions with the N-terminus and ECL2 of CCR5." Virology (2009): Markham, Richard, Wei-Cinn Wang, Anton Weisstein, Zhe Wang, Alvaro Munoz, Alan Templeton, Joseph Margolick, David Vlahov, Homayoon Farzadegan, AND Xiao-Fang Yu. "Patterns of HIV-1 evolution in individuals with differing rates of." Medical Sciences. 95. (1998): 12568–12573.