In situ Analysis of HIV-1 Uncoating
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Transcript of In situ Analysis of HIV-1 Uncoating
In situ Analysis of HIV-1 Uncoating
Omar PerezDepartment of Microbiology and Immunology
College of MedicineUniversity of Illinois at Chicago
Advisor: Thomas J. HopeDepartment of Cell and Molecular Biology
Feinberg School of MedicineNorthwestern University
HIV-1 Life Cycle (Early Phase)
Fluorescent Microscopy based approach
Adapted from Turner and Summers 1999
1. Immediately after entry
2. Enroute to nucleus
3. At nucleus/nuclear pore
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
Campbell and Perez et al. Virology (2006)
Incorporation of Fluorescent Fusion Proteins into HIV-1
Labeling of Viral Membranes With S15-mCherry
Unfused
Fused
1.Allow Pseudotyped HIV to Bind at 17ºC
2.Shift to 37°C to Allow Endocytosis
3.Fix and analyze at various time points
4. Control
Trafficking Studies
GFPVpr+ S15Cherry+ Virion
Virion Incorporated S15-mCherry is Lost Following Productive Entry into Target Cells
Campbell and Perez et al. Virology (2006)
BafA
Virion Incorporated S15-mCherry is Lost Following Productive Entry into Target Cells
Campbell and Perez et al. Virology (2006)
No Drug
% remaining virions %S15+ n
0 hr Baf 84.56+/- 7.5 90.6 +/ 2.3 8990hr 100 +/- NA 90.4 +/-2.2 11741hr 50.7 +/- 5.1 39.2 +/-5 6532hr 31.1 +/- 13.9 23.9 +/-3.4 2794hr 11.5 +/- 3.2 15.8+/-4.4 894hrBaf 108 +/- 38.4 86.77+/-2.9 928
Temporal Analysis of S15-mCherry Loss Following Infection
Hours Post Infection
Campbell and Perez et al. Virology (2006)
S15-mCherry (+)
S15-mCherry (-)
Temporal Analysis of S15-mCherry Loss Following Infection
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1462 1463 1464 1465 14660 1 2 4
Hours Post Infection
% R
emai
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Pop
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Campbell and Perez et al. Virology (2006)
S15-mCherry (+)
S15-mCherry (-)
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
p24CA persists with HIV-1 core following fusion
p24 (+)p24 (-)
p24CA Mutants Used
Forshey et. al. J. Virol 2002
Assay tracks kinetics of uncoating
• Genome expands during RT• Mutations that disrupt p24CA stability are not
infectious• Use RT inhibitor to exam p24CA retention
Is There A Link Between Reverse Transcription and Uncoating?
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1 2 4
Time P.I. (hrs)
% Fused GFPVpr (+) Particles
No DrugNevirapine
Inhibiting RT Prolongs Uncoating
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
AcknowledgementsHope Lab Members
Present:Tom Hope
Edward CampbellKelly Farbach
Scott McCoombeMinh Dinh
Sheila BarryDaniel Gallo
Shetha ShukairAnn Trull
Mike McRavenMeegan Anderson
Jill Kowalski
Past:Jenny AndersonCandace Gomez
Hamani HendersonAndrea Anderson
Marta MelarXialou Wu
Jason LeBlancNicole Byers
Molecular Immunogenetics Program, Oklahoma Medical Research Foundation
William Rodgers
Division of Human Biology, Fred Hutchinson Cancer Research Center, Seattle
WashingtonMichael Emerman
Department of Microbiology and Immunology, Vanderbilt University
School of MedicineChris Aiken
American Society for MicrobiologyRobert D. Watkins Research Fellowship
Inhibiting RT Prolongs Uncoating