1. Real time imaging of HIV uncoating in Living Cells
Joao Mamade and Thomas J. Hope
Northwestern University

2. Models of Uncoating Immediate disassembly of the cone.
Early: Uncoating in the cytoplasm with some retention of CA.
Late: Cores at the Pores.
CA retention after Uncoating
Hulme et al 2015
EARLY: McDonald et al 2002, Hulme et al 2011, Xu et al 2013, Francis et al 2016, others
LATE: Stremlau et al 2006, Arhel et al 2008, Rasayyaah et al 2013, others

3. Leveraging Core Integrity Assay for Live Cell Imaging
Yu et al. J Mol Biol. 425: MA CA NC P6
GFP
Hubner et al., J Virology (Chen lab)
Gag-iGFP construct
Use fluid phase marker for core integrity
fusion
Uncoating?
mCherry-Vpr HIV-iGFP IN-mRuby
Dual labeled virus
iGFP fluid phase marker in Core and “viroplasm”
Observe lost of majority of fluid phase GFP at fusion
Observe loss of remaining GFP as capsid integrity is lost (Uncoating?)

4. iGFP
p24/p55
Lamin-B
10μm
Imaging with a Twist Super low MOI: Long Term Imaging to determine infection
iGFP
p24/p55
Lamin-B
10μm
iGFP
p24
p17-AF405
Lamin-B
iGFP
p24/p55
Lamin-B
10μm

5. That Virus Infected That Cell!
Virions producing infection
Median of dual drop events
25.0 mins
29.3 mins
Number of imaged cells
~290
~4000

6. Models of Uncoating Immediate disassembly of the cone.
Early: Uncoating in the cytoplasm with some retention of CA.
Late: Cores at the Pores.
CA retention after Uncoating
EARLY: McDonald et al 2003, Hulme et al 2011, Xu et al 2013, Francis et al 2016, others
LATE: Stremlau et al 2006, Arhel et al 2008, Rasayyaah et al 2013, others