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

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

Leveraging Core Integrity Assay for Live Cell Imaging Yu et al. J Mol Biol. 425:112-23. 2013 MA CA NC P6 GFP Hubner et al., J Virology. 2007 (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?)

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

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

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