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

0

20

40

60

80

100

1462 1463 1464 1465 14660 1 2 4

Hours Post Infection

% R

emai

ning

Pop

ulati

on

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?

0

10

20

30

40

50

60

70

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