HIV-1 entry and fusion in reporter cells and primary CD4+ T cells is dynamin dependent
Irrespective of the site of entry it is clear that actin rearrangements and dynamin are required for successful infection (Barrero-Villar et al., 2009; Gordon-Alonso et al., 2013). Interestingly, even if several reports show the relevance of dynamin in HIV fusion (Miyauchi et al., 2009; Pritschet et al., 2012; Sloan et al., 2013) its role during this process has not been clarified yet. The dynamin family of GTPases exists in multiple oligomerization forms and is normally found in equilibrium between dimers, tetramers and octamers. Higher order assemblies of dynamin promote its GTPase activity and its oligomerization might be regulated by lipids, SH3-domain containing proteins, microtubules and actin. Actin might be a direct regulator of dynamin oligomerization and in this context lifetime imaging (FLIM) and Number and Brightness have been reported to be able to specifically assess dynamin olygomerization in live cells. Here, we show that the HIVJR-FL kinetics using point inhibitors for fusion (T20) and dynasore (Kirchhausen et al., 2008; Macia et al., 2006) were synchronized when no spinoculation protocols (Guo et al., 2011) were applied using a time-of-addition β-lactamase assay (Cavrois et al., 2002) suggesting a crucial role for dynamin right at the moment of fusion. In contrast, a big delay between dynasore and NH4Cl inhibitors was seen for HIVVSV-G in reporter cells, suggesting that in this case dynamin GTPase activity is important for HIVVSV-G entry, but not necessarily for fusion. We further calculated the oligomeric state of dynamin using advanced lifetime imaging (Padilla-Parra and Tramier, 2012) and Number and Brightness. The oligomeric state of dynamin was found to be much lower for HIVJR-FL fusion than HIV virions pseudotyped with VSV-G (HIVVSV-G)
Date: 13 October 2015, 13:00 (Tuesday, 1st week, Michaelmas 2015)
Venue: Sir William Dunn School of Pathology, South Parks Road OX1 3RE
Venue Details: EPA seminar room
Speaker: Sergi Padillaparra (University of Oxford)
Organising department: Sir William Dunn School of Pathology
Organisers: Rebecca Moore (University of Oxford), Helen Farr (University of Oxford), Joanna Miller (University of Oxford), Eva Gluenz (University of Oxford), Kenny Moore (University of Oxford), Rachel Exley (University of Oxford)
Part of: Bug Sessions in infectious disease
Booking required?: Not required
Audience: Members of the University only
Editor: Rebecca Moore