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A Closer Look at the interaction between CCR5 and HIV

A Closer Look at the interaction between CCR5 and HIV. Atchison RE, Gosling J, Monteclaro FS, Franci C, Diglio L, Charo IF, Goldsmith MA. 1996. Multiple Extracellular Elements of CCR5 and HIV-1 entry: Dissociation from the Response of Chemokines. Science 274: 1924-1926 By: Carolina Valdes.

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A Closer Look at the interaction between CCR5 and HIV

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  1. A Closer Look at the interaction between CCR5 and HIV Atchison RE, Gosling J, Monteclaro FS, Franci C, Diglio L, Charo IF, Goldsmith MA. 1996. Multiple Extracellular Elements of CCR5 and HIV-1 entry: Dissociation from the Response of Chemokines. Science 274: 1924-1926 By: Carolina Valdes

  2. CCR5 Role as a Chemokine Receptor • Cell surface proteins that bind small peptides called chemokines • Results in the recruitment of immune cells to the site of tissue damage or disease • Chemokines: MIP-1, MIP-1, and RANTES • Modulators of HIV-1 replication cycle

  3. Characteristic feature: serpentine seven transmembrane-spanning domain structure • Extracellular portion: chemokine binding • Intracellular portion: cell signalling

  4. Cell Signaling in CCR5 • InsP3 (inositol phosphate)/DAG (diacylglycerol) pathway • second messenger system • Controls various responses – secretion of hormones, cell division, etc • Indicates signal transduction after ligand binding to receptor

  5. Predicted Structure of CCR5

  6. Why CCR5? • Secondary Receptor on CD4+ T lymphocytes • Essential Cofactor for HIV cell entry • Confers susceptibility to infection by certain HIV-1 strains in the presence of CD4+ cells

  7. Purpose • To investigate the structural features of CCR5 that contribute to susceptibility of infection • Compared to murine CCR5 and CCR2B

  8. Procedure • Construction of transient transfection-infection system • cells expressing CD4 receptors with natural chemokine receptor or receptor variants • epitope was placed on NH2 terminus of each chemokine receptor

  9. Transfected cells exposed to Ba-L (Macrophage-tropic HIV-1 strain) • Cell entry measured by Flourescence-Activated Cell Sorting (FACS) • Looked for expression of viral capsid protein p24

  10. Is HIV entry dependent on CCR5? • Expression of CD4 alone proved insufficient for susceptibility to infection • Expression of CD4 and human CCR5 allowed for vigorous infection

  11. Immunoassay of CCR5-dependent cell entry by HIV-1 Ba-L

  12. Evaluation of Murine CCR5 as a Coreceptor • Rationale: • The sequence of murine CCR5 (mCCR5) is similar to the human form (82% amino acid identity) • mCCR5 displayed no detectable capacity to support infection

  13. Naming the Chimeras • Four letters – point out the external domain of exchange • Order: NH2 terminus and extracellular loops 1,2,3. • Example: HMMM- replaces mCCR5 NH2 terminus with corresponding human receptor

  14. Predicted CCR5 Structure

  15. What are the critical elements that mCCR5 lacks? • Chimera of mCCR5 with the NH2-terminal extracellular segment of hCCR5 segment - restored coreceptorfunction (HMMM) • Chimera of hCCR5 containing the NH2-terminusof the mCCR5 - robust coreceptor function (MHHH)

  16. Chimera of mCCR5 with the extracellular loop 1 of hCCR5 – reestablished coreceptor function (MHMM) • Chimera of mCCR5 containing either extracellular loop 2 (MMHM) or loop 2 and 3 (MMHH)– displayed some coreceptor activity

  17. Coreceptor Activity of hCCR5/mCCR5 Chimeras

  18. Evaluation of human CCR2B as a coreceptor for HIV entry • Comparison of hCCR5 and hCCR2B: 2 more distant receptors (71% amino acid identity) with different ligand specificities • Rationale: to determine if exchanges between hCCR5 and hCCR2B can establish coreceptor activity

  19. hCCR2B exhibited no coreceptor activity for Ba-L (2222) • Chimera of CCR2B with the NH2-terminal extracellular segment of hCCR5 segment - robust coreceptorfunction (5222) • Chimera of hCCR5 containing the NH2-terminusof the CCR2B - restoredcoreceptor function (2555)

  20. Chimera representing the NH2-terminal half of CCR2B linked to the COOH-terminal halfof human CCR5– no coreceptor function

  21. Transfection-infection assay results with CCR5/CCR2B chimeras

  22. Signaling Response of CCR5/CCR2B Chimeras • Rationale: • to determine the ligand-binding properties of the chimeras • to evaluate its association to viral coreceptor activity • Note: • CCR2B ligand: MCP-1 • CCR5 ligand: MIP-1

  23. Chimera 2255 transmitted signals in response to both ligands (MCP-1 and MIP-1) • Chimera 5222 exhibited no detectable signaling in response to either ligand.

  24. Signaling responses of CCR5/CCR2 chimeras to chemokine ligands

  25. Conclusions • Expression of CD4 and human CCR5 allowed for vigorous infection

  26. Transfection-infection system of mCCR5/hCCR5 • Both the NH2-terminus and distalportions of the receptor are contributory to HIV-1 coreceptoractivity  BUT neither element alone is essential • multiple elementsdistributed throughout the extracellular segments appear to contributeto viral entry

  27. Transfection-infection system of CCR2B/hCCR5 • Within the overall interaction, accommodations in one or another contributory segment of CCR5 can be made without abolishingentirely coreceptor activity. • viral coreceptor activity is dissociable fromligand-dependent signaling responses

  28. References • Howard OM. Shirakawa A. Turpin JA. Maynard A. Tobin GJ. Carrington M. Oppenheim JJ. Dean M. 1999. Naturally-Occurring CCR5 Extracellular and Transmembrane Domain Variants Affect HIV-1 Coreceptor and Ligand Binding Fuunction. J Biol Chem 274: 16228-16234. • McNicholl JM. Smith DK. Qari SH. Hodge T. 1997. Host Genes and HIV: The Role of the Chemokine Receptor Gene CCR5 and Its Allele (32 CCR5). JAMA 3: 238-249. • O'Brien SJ. Dean M.  1997. In Search of AIDS-Resistant Genes. Scientific American

  29. QUESTIONS?

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