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In situ Analysis of HIV-1 Uncoating

In situ Analysis of HIV-1 Uncoating. Omar Perez Department of Microbiology and Immunology College of Medicine University of Illinois at Chicago. Advisor: Thomas J. Hope Department of Cell and Molecular Biology Feinberg School of Medicine Northwestern University.

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In situ Analysis of HIV-1 Uncoating

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  1. In situ Analysis of HIV-1 Uncoating Omar Perez Department of Microbiology and Immunology College of Medicine University of Illinois at Chicago Advisor: Thomas J. Hope Department of Cell and Molecular Biology Feinberg School of Medicine Northwestern University

  2. 1. Immediately after entry 2. Enroute to nucleus 3. At nucleus/nuclear pore HIV-1 Life Cycle (Early Phase) Fluorescent Microscopy based approach Adapted from Turner and Summers 1999

  3. Productive vs Non-productive Cores • Many cores entering cell non-productive • Intracellular trafficking • Fluorescent dNTPs - authentic RTCs • Cumbersome and disruptive to cell • DiD labeling • Inconsistent labeling efficiency

  4. Incorporation of Fluorescent Fusion Proteins into HIV-1 Campbell and Perez et al. Virology (2006)

  5. Labeling of Viral Membranes With S15-mCherry Unfused Fused

  6. 1.Allow Pseudotyped HIV to Bind at 17ºC 2.Shift to 37°C to Allow Endocytosis 4. Control 3.Fix and analyze at various time points GFPVpr+ S15Cherry+ Virion Trafficking Studies

  7. Virion Incorporated S15-mCherry is Lost Following Productive Entry into Target Cells BafA Campbell and Perez et al. Virology (2006)

  8. Virion Incorporated S15-mCherry is Lost Following Productive Entry into Target Cells No Drug Campbell and Perez et al. Virology (2006)

  9. Temporal Analysis of S15-mCherry Loss Following Infection S15-mCherry (+) S15-mCherry (-) Hours Post Infection Campbell and Perez et al. Virology (2006)

  10. % Remaining Population 0 1 2 4 Hours Post Infection Temporal Analysis of S15-mCherry Loss Following Infection S15-mCherry (+) S15-mCherry (-) Campbell and Perez et al. Virology (2006)

  11. Two Populations of Fused HIV-1 particles Horizontal arrows show GFPVpr(+) p24CA(+) Vertical arrows show GFPVpr(+) p24CA(-) *image is from 1hr time point. Shows 1 Z

  12. p24CA persists with HIV-1 core following fusion p24 (+) p24 (-)

  13. p24CA Mutants Used Forshey et. al. J. Virol 2002

  14. Assay tracks kinetics of uncoating

  15. Is There A Link Between Reverse Transcription and Uncoating? • Genome expands during RT • Mutations that disrupt p24CA stability are not infectious • Use RT inhibitor to exam p24CA retention

  16. Inhibiting RT Prolongs Uncoating

  17. Summary • Developed novel in situ assay • Visualize kinetics of uncoating • p24CA remains associated with core for some time following fusion • In situ results correlates with biochemical data • RT may play a role in p24CA uncoating

  18. Hope Lab Members Present: Tom Hope Edward Campbell Kelly Farbach Scott McCoombe Minh Dinh Sheila Barry Daniel Gallo Shetha Shukair Ann Trull Mike McRaven Meegan Anderson Jill Kowalski Past: Jenny Anderson Candace Gomez Hamani Henderson Andrea Anderson Marta Melar Xialou Wu Jason LeBlanc Nicole Byers Molecular Immunogenetics Program, Oklahoma Medical Research Foundation William Rodgers Division of Human Biology, Fred Hutchinson Cancer Research Center, Seattle Washington Michael Emerman Department of Microbiology and Immunology, Vanderbilt University School of Medicine Chris Aiken American Society for Microbiology Robert D. Watkins Research Fellowship Acknowledgements

  19. Inhibiting RT Prolongs Uncoating

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