Resolution of cell-associated and cell-free HIV-1 viral dynamics

The entry of HIV-1 into target cells and viral spread among cells of the immune system are two criticalevents in the viral life cycle. This study aimed to further characterise HIV-1 pathogenesis three-fold.Firstly, development of fluorescent markers for HIV-1 content and lipid resulted in a novel system togenerate fluorescent HIV-1 particles with near wild-type infectivity for use in single particle imaging.Through stringent analysis of the viral progeny, two key caveats of HIV-1 particle tracking wereidentified. Firstly, the viral content was compartmentalised both within and outside of the HIV-1capsid core. Also, although improvements were made to the lipid label, significant numbers offluorescent microvesicles that co-associate with HIV-1 were recognised. These key findings influencethe interpretation of these particles when used for single particle tracking.Secondly, this fluorescent virus was used to characterise one of the most potent forms of viral spread,transfer across the dendritic cell and CD4+ T cell synapse. In this context, a new pathogenic structurewas identified, which we now term HIV-1 filopodia. These finger-like structures protrude from themembrane and are used by the newly formed virions as a means of cell-cell transfer. The spread ofHIV-1 via viral filopodia was found to be reliant on the viral protein Nef, specifically in the domainthat interacts with the cellular protein Dynamin-II.Finally, using a panel of novel small molecule inhibitors, along with well characterised virologicalmethods, it was investigated whether endocytosis was a pre-requisite for entry into primary cell targetsof HIV-1. Significant post-entry blocks of viral infection were detected when using Dynamin-IIinhibitors. In studies of primary cells, limited inhibition of HIV-1 entry was observed in macrophagesand resting CD4+ T cells, while viral entry was blocked in HeLa-based and activated CD4+ T cells.This thesis provides new, stringent techniques for visualising HIV-1 entry and spread at the cellularlevel. Using this foundation, new viral pathogenic structures we now call HIV-1 filopodia wereidentified. In addition, this foundation has further demonstrated the limits to using single particletracking to dissect viral entry. Given these limitations, an alternative approach was developed to studyHIV-1 entry and it was ultimately observed that endocytosis of HIV-1 is not a pre-requisite for HIV-1fusion and subsequent infection