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Chris Rhodes and Isaiah Castaneda Loyola Marymount University Department of Biology

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 368 11/2/11. Outline.

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Chris Rhodes and Isaiah Castaneda Loyola Marymount University Department of Biology

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  1. 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 368 11/2/11

  2. 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

  3. 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

  4. 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

  5. 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

  6. 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

  7. 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

  8. 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: • GR • RG

  9. 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

  10. 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

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

  12. 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

  13. 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

  14. 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

  15. 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

  16. 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

  17. 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

  18. 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. 400.1 (2009): 145-55. 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.

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